Electroprocessing in Drug Delivery and Cell Encapsulation

ABSTRACT

The invention is directed to novel compositions comprising an electroprocessed material and a substance, their formation and use. The electroprocessed material can, for example, be one or more natural materials, one or more synthetic materials, or a combination thereof. The substance can be one or more therapeutic or cosmetic substances or other compounds, molecules, cells, vesicles. The compositions can be used in substance delivery, including drug delivery within an organism by, for example, releasing substances or containing cells that release substances. The compositions can be used for other purposes, such as prostheses or similar implants.

PRIOR RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applicationSer. No. 60/241,008 filed Oct. 18, 2000, U.S. provisional patentapplication Ser. No. 60/270,118 filed Feb. 22, 2001, U.S.non-provisional patent application Ser. No. 09/714,255 filed Nov. 17,2000, and U.S. non-provisional patent application Ser. No. 09/946,158filed Sep. 4, 2001.

FIELD OF THE INVENTION

The present invention relates to novel compositions comprisingelectroprocessed materials with substances, and methods of making andusing these compositions in delivery of substances.

BACKGROUND OF THE INVENTION

Numerous methods exist for delivering substances to desired locations invivo or in vitro. One such method uses devices or objects that contain asubstance and will release the substance within a desired location. Onedesirable application for such methods is the administration of suchobjects to a location within the body of an organism, followed by thesubsequent release of the desired substance into the body. In theseexamples, the implant often contains the substance and a carrier. Afterimplantation, the substance is released by a variety of means including,for example, diffusion from an implant or dissolution or otherdegradation of a capsule coat.

Biocompatibility is a desirable attribute in compositions designed forsubstance delivery. With surgical and subdermal implants, for example,the substance to be delivered is often contained in a matrix comprisedof synthetic polymers. Where natural products are used in makingbandages, the products typically comprise wood products such ascellulose or other materials that are not readily absorbed by the bodyof the recipient. Accordingly, such bandages must eventually be removed.Implants compressed from natural materials that may be absorbed by thebody are one way to improve biocompatibility and is one area in whichimprovements are desired.

There is also a continuing need for greater versatility and flexibilityin substance delivery technology. Additional techniques for controllingrelease kinetics and spatial patterns of release or delivery areexamples of developments that can improve substance delivery. Implantsin which there is refined control of structure at the microscopic ormolecular level and overall implant shape are also desired. Such methodscould allow, for example, further refinements in control of pore size orother attributes that affect diffusion in and out of a matrix, or morerefined control of the distribution of a substance within a matrix. Newmethods that allow encapsulation of living cells within a matrix areespecially desired. Such methods would allow implants to contain, forexample, cells that produced desired substances, cells that promotetissue growth, or cells that serve both of these functions.

What is needed therefore are new compositions for use in drug deliverythat provide additional and improved methods of controllingconfiguration of drug delivery systems. Compositions with improvedbiocompatibility compared to those currently used in substance deliveryand/or that can contain living cells are also needed. What is furtherneeded are new methods of substance delivery using such compositions.Finally, methods for making such compositions are also needed.

SUMMARY OF THE INVENTION

The present invention seeks to overcome the limitations in the prior artby providing compositions comprising an electroprocessed material and asubstance. The substance may be the material itself, or anothersubstance which may be delivered with the electroprocessed material to adesired site. Sometimes the compositions comprising an electroprocessedmaterial and a substance are in the form of a matrix. Theelectroprocessed materials include any natural or non-natural materialsor blends thereof. The substance is released from the composition orcauses the release of molecules or compounds from the composition.Substance release can occur in vitro, in vivo, or both.

The present invention also includes a method for delivery of substancesto a location using the present compositions comprising anelectroprocessed material and a substance. The locations can be invitro, in vivo, or both. The invention also includes methods for makingthe compositions of the present invention.

The compositions of the present invention include an electroprocessedmaterial and a substance. The material can include naturally occurringmaterials, synthetically manufactured materials, or combinationsthereof. Naturally occurring materials include natural organic orinorganic materials, genetically engineered materials and includesynthetic alterations thereof. Synthetic materials include materialsprepared through any method of artificial synthesis, processing, ormanufacture. The invention includes materials that degrade and canabsorbed by the body, or will persist in whole or in part and becomeportions of an extracellular tissue matrix. The compositions may be madeusing any electroprocessing technique, including but not limited toelectrospinning, electroaerosol, electrospraying or electrosputteringtechniques, or any combination thereof. Accordingly, electroprocesseddroplets, particles, fibers, fibrils, or combinations thereof are allincluded in the compositions of the present invention. In a preferredembodiment, the electroprocessed materials form a matrix, and in somecases are similar to an extracellular matrix. Matrices may also beformed from materials that can combine to form another material, such asprecursor materials. For example, fibrinogen, when combined withthrombin, will form fibrin.

Any material that may be electroprocessed may be used to form anelectroprocessed material to be combined, either before, during or afterelectroprocessing, with a substance, to form the compositions of thepresent invention. The compositions of the present invention contain oneor more substances. The substance includes any type of substancedesired, with examples including molecules, cells, objects, orcombinations thereof. In some cases, the substance is theelectroprocessed material itself. Molecules can be any size, complexity,or type, including both organic or inorganic molecules as well as anycombination of molecules. Molecules include naturally occurring andsynthetic molecules. Examples of molecules include, but are not limitedto therapeutics, cosmetics, nutraceuticals, vitamins, minerals,humectants, molecules produced by cells, including normal cells,abnormal cells, genetically engineered cells and cells modified throughany other process. Both eukaryotic and prokaryotic cells are included inthe category of substances. Substances also include, without limitation,antigens, antimicrobials, antifungals, molecules that can cause acellular or physiological response, metals, gases, minerals, ions, andelectrically, magnetically and electromagnetically (i.e., light)reactive materials. Cells are derived from natural sources or arecultured in vitro. Combinations of different types or categories ofcells can be used. Examples of objects include, but are not limited to,cell fragments, cell debris, organelles and other cellular components,tablets, viruses, vesicles, liposomes, capsules, and other structuresthat serve as an enclosure for molecules. It is to be understood thatthe composition of the present invention comprises at least onesubstance. Accordingly, numerous substances or combinations of similaror different substances may be combined with the electroprocessedmaterial. The substances may be combined with the electroprocessedmaterial through electroprocessing techniques or through othertechniques. The invention also includes embodiments in which thecomposition comprises electroprocessed matrix materials without anadditional substance. In that embodiment, the electroprocessed matrixmaterials may act as a substance.

The invention provides numerous uses for the compositions of the presentinvention. One preferred use is the delivery of substances. Substancedelivery from the compositions of the present invention can occur invivo, for example upon or within the body of a human or animal.Substance delivery can also occur in vitro, for example within a cellculture apparatus or well. Substances delivered include those substancescontained within the compositions, other substances produced by thesubstance contained in the composition, or both. For example, asubstance may be a cell contained within the electroprocessed material,and the cell may synthesize and release one or more molecules. Cells mayrelease molecules in response to signals, so that the molecules arereleased in a specific desired circumstance. For example, an induciblepromoter in an engineered cell within an electroprocessed material maybe used to stimulate the expression and or release of a growth factor.

The compositions of the present invention are versatile with respect tocontrol of substance release from the compositions. Release kinetics ofsubstances can be controlled by manipulating a wide variety of matrixparameters. In various embodiments, the release rate, onset of release,release of more than one compound either at the same or different times,creation of gradients of release and spatial patterns of release may bemanipulated. Compositions that contain electrical or magnetic materialscan be influenced to move, cause motion, or produce a biologicalactivity by applying an electric current or a magnetic field to thecomposition located on or within a body, or in vitro. Electroprocessedcompositions that contain light sensitive components may be designed.These compositions may move or be induced to release or bind substancesin response to specific wavelengths of light. Compositions containingnucleic acids or genetically engineered cells, for example, can be usedin gene therapy. Other examples include embodiments used in wound care,tissue or organ replacements, and prostheses. In some embodiments, theelectroprocessed material itself contains desired properties ofsubstances, and acts as a substance without addition of anothersubstance. The invention thus includes a wide variety of methods ofusing the compositions of the present invention in medical veterinary,agricultural, research and other applications. The compositions of thepresent invention provide safer and more predictable release ofsubstances and provide a major advance in the field of substancedelivery, especially drug delivery.

The invention also includes methods for making the compositions of thepresent invention using any type of electroprocessing technique,combination of electroprocessing techniques, or a combination of anelectroprocessing technique and another technique, such as aerosoltechniques. The method includes streaming, spraying, dropping orprojecting one or more solutions, fibers, or suspensions comprising thematerials to be electroprocessed toward a target under conditionseffective to deposit the materials on a substrate. The substances to becombined with the electroprocessed materials may be electroprocessedtoward the target either before, during or after electroprocessing thematerial. In this manner, the substance may be incorporated within theelectroprocessed material during formation, or may coat theelectroprocessed material. Accordingly, one or a plurality of sources ofmaterials and substances is used to provide the ingredients for theelectroprocessed composition of the present invention. For example,collagen and a polymer such as poly glycolic acid may beelectroprocessed through any combination of electrospinning andelectrospraying from two sources. At the same time or at selected timesthereafter, substances may be provided from other sources: for example,a third source provides a growth factor, a fourth source provides ananti-angiogenic factor, and a fifth source provides genetically alteredfibroblasts. These sources of substances may provide the substancesthrough one or more electroprocessing techniques, such as electrospin,electrospray, electroaerosol, electrosputter or any combination thereof.These sources may also provide the substances to the electroprocessedmaterial through non-electroprocessing techniques, such as aerosoldelivery, dripping, coating, soaking or other techniques.

In one preferred embodiment, the compositions of the present inventioncomprise one or more electroprocessed materials that form a matrixcombined with at least one substance. Either the source or target ischarged, and the other is grounded. The substrate upon whichelectrodeposition occurs can be the target itself or another object ofany shape or type. For example, the substrate can be an object disposedbetween the orifice and the target. In one embodiment, the substrate isa location on or within an organism, such as a tissue, a wound site, adesired location for substance delivery, or a surgical field in whichthe composition is to be applied. By manipulating process parameters,compositions of the present invention can be manufactured with apredetermined shape, for example, for depositing the material onto orinto a molded substrate. Substrate shape can be manipulated to achieve aspecific three-dimensional structure. Targets can also be rotated orotherwise moved or manipulated during electroprocessing to controldistribution of the electroprocessed material and, in embodimentsinvolving electroprocessed fibers, the orientation of the fibers.Substances included in the composition can be combined with the matrixmaterial by any means before, during, and/or after electrodeposition.

The electroprocessed compositions may be formed into any desired shape.For purposes of substance delivery, the desired shape is dictated by theapplication. Non-limiting examples include the following: in the form ofa patch for application to the skin; in the form of a wafer or tabletfor ingestion; in the form of a wafer for application to a site ofremoval of a glioma; in the form of a wrap to surround a tumor; in aparticulate form for spraying on a surgical site; and in a particulateform for delivery of substances through inhalation.

Accordingly, it is an object of the present invention to overcome theforegoing limitations and drawbacks by providing compositions comprisingan electroprocessed material and a substance.

Another object of the present invention is to provide compositionscomprising an electroprocessed natural material and a substance.

Yet another object of the present invention is to provide compositionscomprising an electroprocessed synthetic material and a substance.

Still another object of the present invention is to provide compositionscomprising blends of an electroprocessed natural material, anelectroprocessed synthetic material and a substance.

Another object of the present invention is to provide compositionscomprising an electroprocessed synthetic material and a substance.

It is an object of the present invention to provide compositionscomprising an electroprocessed material and a substance, wherein thesubstances comprises comprising cells.

Another object of the present invention is to provide compositionscomprising an electroprocessed material and a substance, wherein thesubstance comprises an object.

Still another object of the present invention is to provide compositionscomprising an electroprocessed material and a substance, wherein thesubstance comprises a molecule.

Yet another object of the present invention is to provide compositionscomprising an electroprocessed material and a substance, wherein thesubstance comprises a therapeutic molecule.

Another object of the present invention is to provide compositionscomprising an electroprocessed material and substances comprisingcombinations of cells, molecules, and/or objects.

Another object of the present invention is to provide methods fordelivery of a substance to a location, comprising placing thecomposition of the present invention at a desired location.

Still another object of the present invention is to provide methods fordelivery of substances to a location inside or upon the body of a humanor animal.

Yet another object of the present invention is to provide methods forretrieval of substances from a location inside or upon the body of ahuman or animal by bonding such substances.

Yet another object of the present invention is to provide methods fordelivery or retrieval of substances to in vitro locations.

Another object of the present invention is to provide methods fordelivery of drugs in vivo.

Yet another object of the present invention is to provide methods ofadministering gene and or peptide therapy.

Another object of the present invention is to provide methods of proteinor peptide therapy.

Still another object of the present invention is to provide methods ofadministering tissue and organ replacements and prostheses.

Another object of the present invention is to provide methods for makingthe compositions of the present inventions.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of an embodiment of an electroprocessingdevice including the electroprocessing equipment and a rotating wallbioreactor.

FIG. 2 is a schematic drawing of an embodiment of an electroprocessingdevice including the electroprocessing equipment and a rotating wallbioreactor.

FIG. 3 is a graph showing the release profile of vascular endothelialgrowth factor (VEGF) from one embodiment of the present inventionobtained by electrospinning a solution comprising collagen, polylacticacid (PLA), polyglycolic acid (PGA), and VEGF.

FIG. 4 is a graph showing the release profile of VEGF from an embodimentof the present invention obtained by electrospinning a solutioncomprising collagen, polylactic acid, and polyglycolic acid, and VEGFand subsequently cross-linking the electroprocessed material by exposureto glutaraldehyde vapor.

FIG. 5 is a graph showing the release profile of tetracycline fromseveral embodiments of the present invention obtained by electrospinningsolutions containing tetracycline along with PLA, poly(ethylene-co-vinylacetate) or a combination of PLA and poly(ethylene-co-vinyl acetate).

FIG. 6 is a graph comparing the release profile of tetracycline from anembodiment of the present invention and several other compositions. Theembodiment of the present invention was obtained by electrospinning asolution containing tetracycline with poly(ethylene-co-vinyl acetate).The other compositions were periodontal fibers containing 25 wt %tetracycline hydrochloride and films containing tetracycline withpolylactic acid, poly(ethylene-co-vinyl acetate) or a combination ofpolylactic acid and poly(ethylene-co-vinyl acetate).

FIG. 7 is a graph showing the release profile of tetracycline fromseveral embodiments of the present invention obtained by electrospinningsolutions containing tetracycline with poly(ethylene-co-vinyl acetate).

FIG. 8 is a schematic drawing of another embodiment of anelectroprocessing device including the electroprocessing equipment and arotating wall bioreactor.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The term “substance” shall be used throughout this application in itsbroadest definition. The term substance includes one or more molecules,objects, or cells of any type or size, or combinations thereof.Substances can be in any form including, but not limited to solid,semisolid, wet or dry mixture, gas, solution, suspension, combinationsthereof. Substances include molecules of any size and in anycombination. Cells include all cell types of prokaryotic and eukaryoticcells, whether in natural state or altered by genetic engineering or anyother process. Cells can be from a natural source or cultured in vitroand can be living or dead. Combinations of different types of cells canbe used. Objects can be of any size, shape, and composition that may becombined with or coupled to an electroprocessed material. Examples ofobjects include, but are not limited to, cell fragments, cell debris,fragments of cell walls, fragments of viral walls, organelles and othercell components, tablets, viruses, vesicles, liposomes, capsules,nanoparticulates, and other structures that serve as an enclosure formolecules. The compositions of the present invention may comprise onesubstance or any combination of substances.

The terms “electroprocessing” and “electrodeposition” shall be definedbroadly to include all methods of electrospinning, electrospraying,electroaerosoling, and electrosputtering of materials, combinations oftwo or more such methods, and any other method wherein materials arestreamed, sprayed, sputtered or dripped across an electric field andtoward a target. The electroprocessed material can be electroprocessedfrom one or more grounded reservoirs in the direction of a chargedsubstrate or from charged reservoirs toward a grounded target.“Electrospinning” means a process in which fibers are formed from asolution or melt by streaming an electrically charged solution or meltthrough an orifice. “Electroaerosoling” means a process in whichdroplets are formed from a solution or melt by streaming an electricallycharged polymer solution or melt through an orifice. The termelectroprocessing is not limited to the specific examples set forthherein, and it includes any means of using an electrical field fordepositing a material on a target.

The term “material” refers to any compound, molecule, substance, orgroup or combination thereof that forms any type of structure or groupof structures during or after electroprocessing. Materials includenatural materials, synthetic materials, or combinations thereof.Naturally occurring organic materials include any substances naturallyfound in the body of plants or other organisms, regardless of whetherthose materials have or can be produced or altered synthetically.Synthetic materials include any materials prepared through any method ofartificial synthesis, processing, or manufacture. Preferably thematerials are biologically compatible materials.

One class of synthetic materials, preferably biologically compatiblesynthetic materials, comprises polymers. Such polymers include but arenot limited to the following: poly(urethanes), poly(siloxanes) orsilicones, poly(ethylene), poly(vinyl pyrrolidone), poly(2-hydroxy ethylmethacrylate), poly(N-vinyl pyrrolidone), poly(methyl methacrylate),poly(vinyl alcohol), poly(acrylic acid), polyacrylamide,poly(ethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylicacid), polylactides (PLA), polyglycolides (PGA),poly(lactide-co-glycolides) (PLGA), polyanhydrides, and polyorthoestersor any other similar synthetic polymers that may be developed that arebiologically compatible. The term “biologically compatible, syntheticpolymers” shall also include copolymers and blends, and any othercombinations of the forgoing either together or with other polymersgenerally. The use of these polymers will depend on given applicationsand specifications required. A more detailed discussion of thesepolymers and types of polymers is set forth in Brannon-Peppas, Lisa,“Polymers in Controlled Drug Delivery,” Medical Plastics andBiomaterials, November 1997, which is incorporated by reference as ifset forth fully herein.

“Materials” also include electroprocessed materials that are capable ofchanging into different materials during or after electroprocessing. Forexample, the protein fibrinogen, when combined with thrombin, formsfibrin. Fibrinogen or thrombin that are electroprocessed as well as thefibrin that later forms are included within the definition of materials.Similarly, procollagen will form collagen when combined with procollagenpeptidase. Procollagen, procollagen peptidase, and collagen are allwithin the definition.

In a preferred embodiment, the electroprocessed materials form a matrix.The term “matrix” refers to any structure comprised of electroprocessedmaterials. Matrices are comprised of fibers, or droplets of materials,or blends of fibers and droplets of any size or shape. Matrices aresingle structures or groups of structures and can be formed through oneor more electroprocessing methods using one or more materials. Matricesare engineered to possess specific porosities. Substances may bedeposited within, or anchored to or placed on matrices. Cells aresubstances which may be deposited within or on matrices.

One preferred class of materials for electroprocessing to make thecompositions of the present invention comprises proteins. Extracellularmatrix proteins are a preferred class of proteins in the presentinvention. Examples include but are not limited to collagen, fibrin,elastin, laminin, and fibronectin. Additional preferred materials areother components of the extracellular matrix, for example proteoglycans.In each case, those names are used throughout the present application intheir broadest definition. There are multiple types of each of theseproteins that are naturally-occurring as well as types that can be orare synthetically manufactured or produced by genetic engineering. Forexample, collagen occurs in many forms and types. All of these types andsubsets are encompassed in the use of the proteins named herein. Theterm protein further includes, but is not limited to, fragments,analogs, conservative amino acid substitutions, and substitutions withnon-naturally occurring amino acids with respect to each named protein.The term “residue” is used herein to refer to an amino acid (D or L) oran amino acid mimetic that is incorporated into a protein by an amidebond. As such, the amino acid may be a naturally occurring amino acidor, unless otherwise limited, may encompass known analogs of naturalamino acids that function in a manner similar to the naturally occurringamino acids amino acid mimetics). Moreover, an amide bond mimeticincludes peptide backbone modifications well known to those skilled inthe art.

Furthermore, one of skill will recognize that, as mentioned above,individual substitutions, deletions or additions which alter, add ordelete a single amino acid or a small percentage of amino acids(typically less than 5%, more typically less than 1%) in an encodedsequence are conservatively modified variations where the alterationsresult in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following six groupseach contain amino acids that are conservative substitutions for oneanother:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

It is to be understood that the term protein, polypeptide or peptidefurther includes fragments that may be 90 to 95% of the entire aminoacid sequence, and also extensions to the entire amino acid sequencethat are 5% to 10% longer than the amino acid sequence of the protein,polypeptide or peptide.

When peptides are relatively short in length (i.e., less than about 50amino acids), they are often synthesized using standard chemical peptidesynthesis techniques. Solid phase synthesis in which the C terminalamino acid of the sequence is attached to an insoluble support followedby sequential addition of the remaining amino acids in the sequence is apreferred method for the chemical synthesis of the antigenic epitopesdescribed herein. Techniques for solid phase synthesis are known tothose skilled in the art.

Alternatively, the proteins or peptides that may be electroprocessed aresynthesized using recombinant nucleic acid methodology. Generally, thisinvolves creating a nucleic acid sequence that encodes the peptide orprotein, placing the nucleic acid in an expression cassette under thecontrol of a particular promoter, expressing the peptide or protein in ahost, isolating the expressed peptide or protein and, if required,renaturing the peptide or protein. Techniques sufficient to guide one ofskill through such procedures are found in the literature.

When several desired protein fragments or peptides are encoded in thenucleotide sequence incorporated into a vector, one of skill in the artwill appreciate that the protein fragments or peptides may be separatedby a spacer molecule such as, for example, a peptide, consisting of oneor more amino acids. Generally, the spacer will have no specificbiological activity other than to join the desired protein fragments orpeptides together, or to preserve some minimum distance or other spatialrelationship between them. However, the constituent amino acids of thespacer may be selected to influence some property of the molecule suchas the folding, net charge, or hydrophobicity. Nucleotide sequencesencoding for the production of residues which may be useful inpurification of the expressed recombinant protein may be built into thevector. Such sequences are known in the art. For example, a nucleotidesequence encoding for a poly histidine sequence may be added to a vectorto facilitate purification of the expressed recombinant protein on anickel column.

Once expressed, recombinant peptides, polypeptides and proteins can bepurified according to standard procedures known to one of ordinary skillin the art, including ammonium sulfate precipitation, affinity columns,column chromatography, gel electrophoresis and the like. Substantiallypure compositions of about 50 to 99% homogeneity are preferred, and 80to 95% or greater homogeneity are most preferred for use as therapeuticagents.

Also, molecules capable of forming some of the named proteins can bemixed with other polymers during electroprocessing to obtain desiredproperties for uses of the formed protein in the matrix.

Throughout this application the term “solution” is used to describe theliquid in the reservoirs of the electroprocessing method. The term isdefined broadly to include any liquids that contain materials to beelectroprocessed. It is to be understood that any solutions capable offorming a material during electroprocessing are included within thescope of the present invention. In this application, the term “solution”also refers to suspensions or emulsions containing the material oranything to be electrodeposited. “Solutions” can be in organic orbiologically compatible forms. This broad definition is appropriate inview of the large number of solvents or other liquids and carriermolecules, such as polyethylene glycol (PEG), that can be used in themany variations of electroprocessing. In this application, the term“solution” also refers to melts, hydrated gels and suspensionscontaining the materials, substances or anything to be electrodeposited.

Solvents

Any solvent that will allows delivery of the material or substance tothe orifice or tip of a syringe under such conditions that the materialor substance will be processed as desired may be used for dissolving orsuspending the material or the substance to be electroprocessed.Solvents useful for dissolving or suspending a material or a substancewill depend on the material or substance. Electrospinning techniquesoften require more specific solvent conditions. For example, noncross-linked fibrin monomer can be electrodeposited or electrospun fromsolvents such as urea, monochloroacetic acid, water,2,2,2-trifluoroethanol, or 1,1,1,3,3,3-hexafluoro-2-propanol (also knownas hexafluoroisopropanol or HFIP). Collagen can be electrodeposited as asolution or suspension in water, 2,2,2-trifluoroethanol, or HFIP.Elastin can be electrodeposited as a solution or suspension in water,2,2,2-trifluoroethanol, isopropanol, or HFIP. Other lower orderalcohols, especially halogenated alcohols, may be used. Proteins andpeptides associated with membranes are often hydrophobic and thus cannotdissolve in aqueous solutions. Such proteins can be dissolved in organicsolvents such as methanol, chloroform, and trifluoroethanol (TFE). Anyother solvents known to one of skill in the protein chemical art may beused, for example solvents useful in chromatography, especially highperformance liquid chromatography. Proteins and peptides are alsosoluble, for example, in HFIP, hexafluoroacetone, chloroalcohols inconjugation with aqueous solutions of mineral acids, dimethylacetamidecontaining 5% lithium chloride, in very dilute acids such as acetic acidand formic acid. N-methyl morpholine-N-oxide is another solvent that canbe used with many polypeptides.

In functional terms, solvents used for electroprocessing have theprincipal role of creating a mixture with a polymer, or polymers, suchthat electroprocessing is feasible. The concentration of a given solventis often an important consideration in determining the type ofelectroprocessing that will occur. For example, in electrospraying, thesolvent should assist in the dispersion of droplets of electroprocessedmaterial so that the initial jet of liquid disintegrates into droplets.Accordingly, solvents used in electrospraying should not create forcesthat will stabilize an unconfined liquid column. In electrospinning,interactions between molecules of electroprocessed material stabilizethe jet, leading to fiber formation. Accordingly, for electrospunembodiments, the solvent should sufficiently dissolve or disperse thepolymer to prevent the jet from disintegrating into droplets and shouldthereby allow formation of a stable jet in the form of a fiber. In someembodiments, the transition from electrospraying to electrospinning canbe determined by examining Brookfield viscosity measurements for polymersolutions as a function of concentration. Brookfield viscosity increasesas concentration of a polymer or other material to be electroprocessedincreases. Above a critical concentration associated with extensivechain entanglements of materials, however, the Brookfield viscosity willincrease more rapidly with concentration, as opposed to a more gradual,linear rise with concentration at lower concentrations. For example, theBrookfield viscosity of a poly(lactide) sample obtained from Alkermesdissolved in chloroform shows an upturn in the Brookfieldviscosity/concentration plot at approximately 7-8% w/v. A sample ofpoly(ethylene-co-vinyl acetate) from Dupont (ELVAX 40W) shows an upturnat 14-15% w/v. In both cases, these departures from linearityapproximately coincide with the transition from electrospraying toelectrospinning.

Compositions of the Present Invention

The Electroprocessed Material

One component of the compositions of the present invention is theelectroprocessed material. As defined above, the electroprocessedmaterial of the present invention can include natural materials,synthetic materials, or combinations thereof. Examples include but arenot limited to amino acids, peptides, denatured peptides such as gelatinfrom denatured collagen, polypeptides, proteins, carbohydrates, lipids,nucleic acids, glycoproteins, lipoproteins, glycolipids,glycosaminoglycans, and proteoglycans.

Some preferred materials are naturally occurring extracellular matrixmaterials and blends of naturally occurring extracellular matrixmaterials, including but not limited to collagen, fibrin, elastin,laminin, fibronectin, hyaluronic acid, chondroitin 4-sulfate,chondroitin 6-sulfate, dermatan sulfate, heparin sulfate, heparin, andkeratan sulfate, and proteoglycans. These materials may be isolated fromhumans or other animals or cells or synthetically manufactured. Someespecially preferred natural matrix materials are collagen and fibrinand fibronectin. Also included are crude extracts of tissue,extracellular matrix material, extracts of non-natural tissue, orextracellular matrix materials (i.e. extracts of cancerous tissue),alone or in combination. Extracts of biological materials, including butnot limited to cells, tissues, organs, and tumors may also beelectroprocessed. Collagen has been electrospun to produce a repeating,banded pattern observed with electron microscopy. This banded pattern istypical of collagen fibrils produced by natural processes (i.e. bandedpattern is observed in collagen when it is produced by cells). In someembodiments, collagen is electrospun such that it has a 65 nm bandingpattern.

It is to be understood that these electroprocessed materials may becombined with other materials and/or substances in forming thecompositions of the present invention. For example, an electroprocessedpeptide may be combined with an adjuvant to enhance immunogenicity whenimplanted subcutaneously. As another example, an electroprocessedcollagen matrix, containing cells, may be combined with anelectroprocessed biologically compatible polymer and growth factors tostimulate growth and division of the cells in the collagen matrix.

Synthetic materials include any materials prepared through any method ofartificial synthesis, processing, or manufacture. The syntheticmaterials are preferably biologically compatible for administration invivo or in vivo. Such polymers include but are not limited to thefollowing: poly(urethanes), poly(siloxanes) or silicones,poly(ethylene), poly(vinyl pyrrolidone), poly(2-hydroxy ethylmethacrylate), poly(N-vinyl pyrrolidone), poly(methyl methacrylate),poly(vinyl alcohol), poly(acrylic acid), polyacrylamide,poly(ethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylicacid), polylactic acid (PLA), polyglycolic acids (PGA),poly(lactide-co-glycolides) (PLGA), nylons, polyamides, polyanhydrides,poly(ethylene-co-vinyl alcohol) (EVOH), polycaprolactone, poly(vinylacetate) (PVA), polyvinylhydroxide, poly(ethylene oxide) (PEO) andpolyorthoesters or any other similar synthetic polymers that may bedeveloped that are biologically compatible. Some preferred syntheticmatrix materials include PLA, PGA, copolymers of PLA and PGA,polycaprolactone, poly(ethylene-co-vinyl acetate), (EVOH), PVA, and PEO.Matrices can be formed of electrospun fibers, electroaerosol,electrosprayed, or electrosputtered droplets, or a combination of theforegoing.

In embodiments in which natural materials are used, those materials canbe derived from a natural source, synthetically manufactured, ormanufactured by genetically engineered cells. For example, geneticallyengineered proteins can be prepared with specific desired sequences ofamino acids that differ from the natural proteins. In one illustrativeembodiment, desirable sequences that form binding sites on a collagenprotein for cells or peptides can be included in higher amounts thanfound in natural collagen.

By selecting different materials, or combinations thereof, manycharacteristics of the electroprocessed material can be manipulated. Theproperties of the matrix comprised of electroprocessed material and asubstance may be adjusted. As discussed in greater detail below,electroprocessed materials themselves can provide a therapeutic effectwhen applied. In addition, selection of matrix materials can affect thepermanency of an implanted matrix. For example, matrices made of fibrinwill degrade more rapidly while matrices made of collagen are moredurable and synthetic matrix materials are more durable still. Use ofmatrices made of natural materials such as proteins also minimizerejection or immunological response to an implanted matrix. Accordinglyselection of materials for electroprocessing and use in substancedelivery is influenced by the desired use. In one embodiment, a skinpatch of electroprocessed fibrin or collagen combined with healingpromoters and anti-rejection substances may be applied to the skin andmay subsequently dissolve into the skin. In another embodiment, animplant for delivery to bone may be constructed of materials useful forpromoting bone growth, osteoblasts and hydroxyapatite, and may bedesigned to endure for a prolonged period of time.

Synthetic components, such as biocompatible substances can be used tomodulate the release of materials from an electroprocessed composition.For example, a drug, or series of drugs or other materials to bereleased in a controlled fashion can be electroprocessed into a seriesof layers. One layer is composed of PGA plus a drug, the next layer PLAplus a drug, a third layer is composed of polycaprolactone plus a drug.The layered construct can be implanted, and as the successive layersdissolve or breakdown, the drug (or drugs) is released in turn as eachsuccessive layer erodes. Unlayered structures can also be used, andrelease is controlled by the relative stability of each component of theconstruct. Another advantage of the synthetic materials is thatdifferent solvents can be used. This can be important for the deliveryof some materials. For example, a drug may be soluble in some organics,and using synthetics increases the number of materials that can beelectroprocessed. The breakdown of these synthetic materials can betailored and regulated in ways that are not available to naturalmaterials. The synthetics are usually not subject to enzymaticbreakdown, and many spontaneously undergo hydrolysis. In addition tothese characteristics, substances can be released from electroprocessedmaterials in response to electrical, magnetic and light based signals.Polymers that are sensitive to such signals can be used, or the polymersmay be derivatized in a way to provide such sensitivity. Theseproperties provide flexibility in making and using electroprocessedmaterials designed to deliver various substances, in vivo and in vitro.

In some embodiments of the present invention, the electroprocessedmaterial itself provides a therapeutic effect. For example, in someembodiments electroprocessed collagen promotes cellular infiltration anddifferentiation, so an electroprocessed collagen matrix alone assistswith healing. The P-15 site, a 15 amino acid sequence within thecollagen molecule, promotes osteoblasts to produce and to secretehydroxyapatite, a component of bone. Another example of specific sitesand sequences within collagen molecules that can be manipulated andprocessed in a similar fashion includes the RGD binding sites of theintegrin molecule. The RGD site is a sequence of three amino acids(Arg-Gly-Asp) present in many matrix materials that serves as a bindingsite for cell adhesion. It is recognized and bound, for example, byintegrins. In addition, electroprocessed materials can be enriched withspecific desired sequences before, during, or after electroprocessing.Sequences can be added in linear or other forms. In some embodiments,the RGD sequences are arranged in a cyclic form referred to as cycloRGD.

An electroprocessed material, such as a matrix, can also be composed ofspecific subdomains of a matrix constituent and can be prepared with asynthetic backbone that can be derivatized. For example, the RGD peptidesequence, and/or a heparin binding domain and/or other sequences, can bechemically coupled to synthetic materials. The synthetic polymer withthe attached sequence or sequences can be electroprocessed into aconstruct. This produces a matrix with unique properties. In theseexamples the RGD site provides a site for cells to bind to and interactwith the matrix. The heparin-binding site provides a site for theanchorage of peptide growth factors to the synthetic backbone.Angiogenic peptides, genetic material, growth factors, cytokines,enzymes and drugs are other non-limiting examples of substances that canbe attached to the backbone of an electroprocessed material to providefunctionality. Peptide side chains may also be used to attach moleculesto functional groups on polymeric backbones. Molecules and othersubstances can be attached to a material to be electroprocessed by anytechnique known in the art.

Another embodiment of matrix materials that have a therapeutic effect iselectroprocessed fibrin. Fibrin matrix material assists in arrest ofbleeding. Fibrin is a component of the provisional matrix that is laiddown during the early stages of healing and may also promote the growthof vasculature in adjacent regions, and in many other ways is a naturalhealing promoter. Fibrinogen as an electroprocessed material can alsoassist in healing. When placed in contact with a wound, for example,fibrinogen will react with thrombin present in the blood plasma from thewound and form fibrin, thereby providing the same healing properties ofa fibrin material.

Substances

As discussed above, the word “substance” in the present invention isused in its broadest definition. In embodiments in which thecompositions of the present invention comprise one or more substances,substances can include any type or size of molecules, cells, objects orcombinations thereof. The compositions of the present invention maycomprise one substance or any combination of substances.

In embodiments in which the substances are molecules, any molecule canbe used. Molecules may, for example, be organic or inorganic and may bein a solid, semisolid, liquid, or gas phase. Molecules may be present incombinations or mixtures with other molecules, and may be in solution,suspension, or any other form. Examples of classes of molecules that maybe used include human or veterinary therapeutics, cosmetics,nutraceuticals, agriculturals such as herbicides, pesticides andfertilizers, vitamins, amino acids, peptides, polypeptides, proteins,carbohydrates, lipids, nucleic acids, glycoproteins, lipoproteins,glycolipids, glycosaminoglycans, proteoglycans, growth factors,hormones, neurotransmitters, pheromones, chalones, prostaglandins,immunoglobulins, monokines and other cytokines, humectants, metals,gases, minerals, ions, electrically and magnetically reactive materials,light sensitive materials, anti-oxidants, molecules that may bemetabolized as a source of cellular energy, antigens, and any moleculesthat can cause a cellular or physiological response. Any combination ofmolecules can be used as well as agonists or antagonists.

Several preferred embodiments use therapeutic molecules include use ofany therapeutic molecule including, without limitation, anypharmaceutical or drug. Examples of pharmaceuticals include, but are notlimited to, anesthetics, hypnotics, sedatives and sleep inducers,antipsychotics, antidepressants, antiallergics, antianginals,antiarthritics, antiasthmatics, antidiabetics, antidiarrheal drugs,anticonvulsants, antigout drugs, antihistamines, antipruritics, emetics,antiemetics, antispasmondics, appetite suppressants, neuroactivesubstances, neurotransmitter agonists, antagonists, receptor blockersand reuptake modulators, beta-adrenergic blockers, calcium channelblockers, disulfarim and disulfarim-like drugs, muscle relaxants,analgesics, antipyretics, stimulants, anticholinesterase agents,parasympathomimetic agents, hormones, anticoagulants, antithrombotics,thrombolytics, immunoglobulins, immunosuppressants, hormoneagonists/antagonists, vitamins, antimicrobial agents, antineoplastics,antacids, digestants, laxatives, cathartics, antiseptics, diuretics,disinfectants, fungicides, ectoparasiticides, antiparasitics, heavymetals, heavy metal antagonists, chelating agents, gases and vapors,alkaloids, salts, ions, autacoids, digitalis, cardiac glycosides,antiarrhythmics, antihypertensives, vasodilators, vasoconstrictors,antimuscarinics, ganglionic stimulating agents, ganglionic blockingagents, neuromuscular blocking agents, adrenergic nerve inhibitors,anti-oxidants, vitamins, cosmetics, anti-inflammatories, wound careproducts, antithrombogenic agents, antitumoral agents, antithrombogenicagents, antiangiogenic agents, anesthetics, antigenic agents, woundhealing agents, plant extracts, growth factors, emollients, humectants,rejection/anti-rejection drugs, spermicides, conditioners, antibacterialagents, antifungal agents, antiviral agents, antibiotics, tranquilizers,cholesterol-reducing drugs, antitussives, histamine-blocking drugs,monoamine oxidase inhibitor. All substances listed by the U.S.Pharmacopeia are also included within the substances of the presentinvention.

Antibiotics useful in the present invention include, but are not limitedto, amoxicillin, amphotericin, ampicillin, bacitracin, beclomethasone,benzocaine, betamethasone, biaxin, cephalosporins, chloramphenicol,ciprofloxacin, clotrimazole, cyclosporin, docycline, enoxacin,erythromycin, gentamycin, miconazole, neomycin, norfloxacin, nystatin,ofloxacin, pefloxacin, penicillin, pentoxifylline,phenoxymethylpenicillin, polymixin, rifampicin, tetracycline, tobrmycin,triclosan, vancomycin, zithromax, derivatives, metabolites, and mixturesthereof, or compounds having similar antimicrobial activity.

Some specific examples of pharmaceutical agents that are useful assubstances include, but are not limited to, quinolones, such as oxolinicacid, norfloxacin, and nalidixic acid, sulfonamides, nonoxynol 9,fusidic acid, cephalosporins, cyclosporine, acebutolol, acetylcysteine,acetylsalicylic acid, acyclovir, AZT, alprazolam, alfacalcidol,allantoin, allopurinol, ambroxol, amikacin, amiloride, aminoacetic acid,aminodarone, amitriptyline, amlodipine, ascorbic acid, aspartame,astemizole, atenolol, benserazide, benzalkonium hydrochloride, benzoicacid, bezafibrate, biotin, biperiden, bisoprolol, bromazepam,bromhexine, bromocriptine, budesonide, bufexamac, buflomedil, buspirone,caffeine, camphor, captopril, carbamazepine, carbidopa, carboplatin,cefachlor, cefalexin, cefatroxil, cefazolin, cefixime, cefotaxime,ceftazidime, ceftriaxone, cefuroxime, selegiline, chloramphenicol,chlorpheniramine, chlortalidone, choline, cilastatin, cimetidine,cisapride, cisplatin, clarithromycin, clavulanic acid, clomipramine,clozapine, clonazepam, clonidine, codeine, cholestyramine, cromoglycicacid, cyanocobalamin, cyproterone, desogestrel, dexamethasone,dexpanthenol, dextromethorphan, dextropropoxiphen, diazepam, diclofenac,digoxin, dihydrocodeine, dihydroergotamine, dihydroergotoxin, diltiazem,diphenhydramine, dipyridamole, dipyrone, disopyramide, domperidone,dopamine, doxycycline, enalapril, ephedrine, epinephrine,ergocalciferol, ergotamine, erythromycin, estradiol, ethinylestradiol,etoposide, Eucalyptus globulus, famotidine, felodipine, fenofibrate,fenoterol, fentanyl, flavin mononucleotide, fluconazole, flunarizine,fluorouracil, fluoxetine, flurbiprofen, furosemide, gallopamil,gemfibrozil, Gingko biloba, glibenclamide, glipizide, Glycyrrhizaglabra, grapefruit seed extract, grape seed extract griseofulvin,guaifenesin, haloperidol, heparin, hyaluronic acid, hydrochlorothiazide,hydrocodone, hydrocortisone, hydromorphone, ipratropium hydroxide,ibuprofen, imipenem, indomethacin, iohexyl, iopamidol, isosorbidedinitrate, isosorbide mononitrate, isotretinoin, ketotifen,ketoconazole, ketoprofen, ketorolac, labetalol, lactulose, lecithin,levocarnitine, levodopa, levoglutamide, levonorgestrel, levothyroxine,lidocaine, lipase, imipramine, lisinopril, loperamide, lorazepam,lovastatin, medroxyprogesterone, menthol, methotrexate, methyldopa,methylprednisolone, metoclopramide, metoprolol, miconazole, midazolam,minocycline, minoxidil, misoprostol, morphine, N-methylephedrine,naftidrofuryl, naproxen, nicardipine, nicergoline, nicotinamide,nicotine, nicotinic acid, nifedipine, nimodipine, nitrazepam,nitrendipine, nizatidine, norethisterone, norfloxacin, norgestrel,nortriptyline, omeprazole, ondansetron, pancreatin, panthenol,pantothenic acid, paracetamol, phenobarbital, derivatives, metabolites,and other such compounds have similar activity. Some preferred drugs orcompounds include, but are not limited to, estrogen, androgen,cortisone, and cyclosporin.

Growth factors useful in the present invention include, but are notlimited to, transforming growth factor-α (“TGF-α”), transforming growthfactor-β (“TGF-β”), platelet-derived growth factors (“PDGF”), fibroblastgrowth factors (“FGF”), including FGF acidic isoforms 1 and 2, FGF basicform 2 and FGF 4, 8, 9 and 10, nerve growth factors (“NGF”) includingNGF 2.5s, NGF 7.0s and beta NGF and neurotrophins, brain derivedneurotrophic factor, cartilage derived factor, bone growth factors(BGF), basic fibroblast growth factor, insulin-like growth factor (IGF),vascular endothelial growth factor (VEGF), granulocyte colonystimulating factor (G-CSF), insulin like growth factor (IGF) I and II,hepatocyte growth factor, glial neurotrophic growth factor (GDNF), stemcell factor (SCF), keratinocyte growth factor (KGF), transforming growthfactors (TGF), including TGFs alpha, beta, beta1, beta2, beta3, skeletalgrowth factor, bone matrix derived growth factors, and bone derivedgrowth factors and mixtures thereof.

Cytokines useful in the present invention include, but are not limitedto, cardiotrophin, stromal cell derived factor, macrophage derivedchemokine (MDC), melanoma growth stimulatory activity (MGSA), macrophageinflammatory proteins 1 alpha (MIP-1alpha), 2, 3 alpha, 3 beta, 4 and 5,IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, TNF-α, and TNF-β. Immunoglobulins useful in the presentinvention include, but are not limited to, IgG, IgA, IgM, IgD, IgE, andmixtures thereof. Some preferred growth factors include VEGF (vascularendothelial growth factor), NGFs (nerve growth factors), PDGF-AA,PDGF-BB, PDGF-AB, FGFb, FGFa, and BGF.

Other molecules useful as substances in the present invention includebut are not limited to growth hormones, leptin, leukemia inhibitoryfactor (LIF), tumor necrosis factor alpha and beta, endostatin,thrombospondin, osteogenic protein-1, bone morphogenetic proteins 2 and7, osteonectin, somatomedin-like peptide, osteocalcin, interferon alpha,interferon alpha A, interferon beta, interferon gamma, interferon 1alpha, and interleukins 2, 3, 4, 5 6, 7, 8, 9, 10, 11, 12, 13, 15, 16,17 and 18.

Embodiments involving amino acids, peptides, polypeptides, and proteinsmay include any type or combinations of such molecules of any size andcomplexity. Examples include, but are not limited to structuralproteins, enzymes, and peptide hormones. These compounds can serve avariety of functions. In some embodiments, the matrix may containpeptides containing a sequence that suppresses enzyme activity throughcompetition for the active site. In other applications antigenic agentsthat promote an immune response and invoke immunity can be incorporatedinto a construct.

In substances such as nucleic acids, any nucleic acid can be present.Examples include, but are not limited to deoxyribonucleic acid (DNA),ent-DNA, and ribonucleic acid (RNA). Embodiments involving DNA include,but are not limited to, cDNA sequences, natural DNA sequences from anysource, and sense or anti-sense oligonucleotides. For example, DNA canbe naked (e.g., U.S. Pat. Nos. 5,580,859; 5,910,488) or complexed orencapsulated (e.g., U.S. Pat. Nos. 5,908,777; 5,787,567). DNA can bepresent in vectors of any kind, for example in a viral or plasmidvector. In some embodiments, nucleic acids used will serve to promote orto inhibit the expression of genes in cells inside and/or outside theelectroprocessed matrix. The nucleic acids can be in any form that iseffective to enhance its uptake into cells.

Cells as a Substance

In embodiments in which cells are a substance, any cell can be used.Cells that can be used include, but are not limited to stem cells,committed stem cells, and differentiated cells. Examples of stem cellsthat can be used include but are not limited to embryonic stem cells,bone marrow stem cells and umbilical cord stem cells. Other examples ofcells used in various embodiments include but are not limited to:osteoblasts, myoblasts, neuroblasts, fibroblasts, glioblasts; germcells, hepatocytes, chondrocytes, keratinocytes, smooth muscle cells,cardiac muscle cells, connective tissue cells, epithelial cells,endothelial cells, hormone-secreting cells, cells of the immune system,and neurons. In some embodiments it is unnecessary to pre-select thetype of stem cell that is to be used, because many types of stem cellscan be induced to differentiate in an organ specific pattern oncedelivered to a given organ. For example, a stem cell delivered to theliver can be induced to become a liver cell simply by placing the stemcell within the biochemical environment of the liver. Cells in thematrix can serve the purpose of providing scaffolding or seeding,producing certain compounds, or both.

Embodiments in which the substance comprises cells include cells thatcan be cultured in vitro, derived from a natural source, or produced byany other means. Any natural source of prokaryotic or eukaryotic cellsmay be used. Embodiments in which the matrix is implanted in an organismcan use cells from the recipient, cells from a conspecific donor or adonor from a different species, or bacteria or microbial cells. Cellsharvested from a source and cultured prior to use are also included.

Some embodiments use cells that have been genetically engineered. Theengineering involves programming the cell to express one or more genes,repressing the expression of one or more genes, or both. One example ofgenetically engineered cells useful in the present invention is agenetically engineered cell that makes and secretes one or more desiredmolecules. When electroprocessed matrices comprising geneticallyengineered cells are implanted in an organism, the molecules producedcan produce a local effect or a systemic effect, and can include themolecules identified above as possible substances. Cells can alsoproduce antigenic materials in embodiments in which one of the purposesof the matrix is to produce an immune response. Cells may producesubstances to aid in the following non-inclusive list of purposes:inhibit or stimulate inflammation; facilitate healing; resistimmunorejection; provide hormone replacement; replace neurotransmitters;inhibit or destroy cancer cells; promote cell growth; inhibit orstimulate formation of blood vessels; augment tissue; and to supplementor replace the following tissue, neurons, skin, synovial fluid, tendons,cartilage, ligaments, bone, muscle, organs, dura, blood vessels, bonemarrow, and extracellular matrix.

Genetic engineering can involve, for example, adding or removing geneticmaterial to or from a cell, altering existing genetic material, or both.Embodiments in which cells are transfected or otherwise engineered toexpress a gene can use transiently or permanently transfected genes, orboth. Gene sequences may be full or partial length, cloned or naturallyoccurring.

Substances in the electroprocessed compositions of the present inventionalso comprise objects. Examples of objects include, but are not limitedto, cell fragments, cell debris, organelles and other cell components,tablets, and viruses as well as vesicles, liposomes, capsules,nanoparticles, and other structures that serve as an enclosure formolecules. In some embodiments, the objects constitute vesicles,liposomes, capsules, or other enclosures that contain compounds that arereleased at a time after electroprocessing, such as at the time ofimplantation or upon later stimulation or interaction. In oneillustrative embodiment, transfection agents such as liposomes containdesired nucleotide sequences to be incorporated into cells that arelocated in or on the electroprocessed material or matrix. In otherembodiments, cell fragments or cell debris are incorporated into thematrix. The presence of cell fragments is known to promote healing insome tissues.

Magnetically or electrically reactive materials are also examples ofsubstances that are optionally included within compositions of thepresent invention. Examples of magnetically active materials include butare not limited to carbon black or graphite, carbon nanotubes,ferrofluids (colloidal suspensions of magnetic particles), and variousdispersions of electrically conducting polymers. Ferrofluids containingparticles approximately 10 nm in diameter, polymer-encapsulated magneticparticles about 1-2 microns in diameter, and polymers with a glasstransition temperature below room temperature are particularly useful.Examples of electrically active polymers include, but are not limitedto, electrically conducting polymers such as polyanilines, polypyrrolesand ionically conducting polymers such as sulfonated polyacrylamides arerelated materials.

In other embodiments, some substances in the electroprocessed materialor matrix supplement or augment the function of other substances. Forexample, when the composition comprises cells that express a specificgene, the composition can contain oligonucleotides that are taken up bythe cells and affect gene expression in the cells. Fibronectin isoptionally incorporated into the matrix to increase cellular uptake ofoligonucleotides by pinocytosis.

As discussed in detail above, the electroprocessed material itself canprovide a therapeutic effect. The invention thus includes embodimentsinvolving methods of causing a therapeutic effect through delivery of anelectroprocessed material to a location without incorporating additionalsubstances in the electroprocessed material. Embodiments in which thematrix material alone is delivered as well as those in which othersubstances are included in the matrix are within the scope of thepresent invention.

Methods of Making the Composition

Electroprocessing

The method of making the compositions includes electroprocessing thematerials and optionally electroprocessing the substances. As definedabove, one or more electroprocessing techniques, such as electrospin,electrospray, electroaerosol, electrosputter or any combination thereofmay be employed to make the electroprocessed materials and matrices inthe compositions of the present invention. In the most fundamentalsense, the electroprocessing apparatus for electroprocessing materialincludes a electrodepositing mechanism and a target substrate. Theelectrodepositing mechanism includes a reservoir or reservoirs to holdthe one or more solutions that are to be electroprocessed orelectrodeposited. The reservoir or reservoirs have at least one orificeor nozzle to allow the streaming of the solution from the reservoirs.One or a plurality of nozzles may be configured in an electroprocessingapparatus. If there are multiple nozzles, each nozzle is attached to oneor more reservoirs containing the same or different solutions.Similarly, there can be a single nozzle that is connected to multiplereservoirs containing the same or different solutions. Multiple nozzlesmay be connected to a single reservoir. Because different embodimentsinvolve single or multiple nozzles and/or reservoirs, any referencesherein to one or nozzles or reservoirs should be considered as referringto embodiments involving single nozzles, reservoirs, and relatedequipment as well as embodiments involving plural nozzles, reservoirs,and related equipment. The size of the nozzles can be varied to providefor increased or decreased flow of solutions out of the nozzles. One ormore pumps used in connection with the reservoirs can be used to controlthe flow of solution streaming from the reservoir through the nozzle ornozzles. The pump can be programmed to increase or decrease the flow atdifferent points during electroprocessing. In this invention pumps arenot necessary but provide a useful method to control the rate at whichmaterial is delivered to the electric field for processing. Material canbe actively delivered to the electric field as a preformed aerosol usingdevices such as air brushes, thereby increasing the rate ofelectrodeposition and providing novel combinations of materials. Nozzlesmay be programmed to deliver material simultaneously or in sequence.

The electroprocessing occurs due to the presence of a charge in eitherthe orifices or the target, while the other is grounded. In someembodiments, the nozzle or orifice is charged and the target is shown tobe grounded. Those of skill in the electroprocessing arts will recognizethat the nozzle and solution can be grounded and the target can beelectrically charged. The creation of the electrical field and theeffect of the electrical field on the electroprocessed materials orsubstances that will form the electroprocessed composition.

The target substrate can also be used as a variable feature in theelectroprocessing of materials used to make the electroprocessedcomposition. Specifically, the target can be the actual substrate forthe materials used to make electroprocessed matrix, or electroprocessedmatrix itself is deposited. Alternatively, a substrate can be disposedbetween the target and the nozzles. For instance, a petri dish can bedisposed between a nozzles and a target, and a matrix can be formed inthe dish. Other variations include but are not limited to non-sticksurfaces between the nozzles and target and placing tissues or surgicalfields between the target and nozzles. The target can also bespecifically charged or grounded along a preselected pattern so that thesolution streamed from the orifice is directed into specific directions.The electric field can be controlled by a microprocessor to create anelectroprocessed matrix having a desired geometry. The target and thenozzle or nozzles can be engineered to be movable with respect to eachother thereby allowing additional control over the geometry of theelectroprocessed matrix to be formed. The entire process can becontrolled by a microprocessor that is programmed with specificparameters that will obtain a specific preselected electroprocessedmatrix. It is to be understood that any electroprocessing technique maybe used, alone or in combination with another electroprocessingtechnique, to make the compositions of the present invention.

Any material that can be electroprocessed is within the method of thepresent invention. Forms of electroprocessed collagen include but arenot limited to preprocessed collagen in a liquid suspension or solution,gelatin, particulate suspension, or hydrated gel. An example for fibrinis a preformed gel electroprocessed by subjecting it to pressure, forexample by using a syringe or airbrush apparatus with a pressure headbehind it to extrude the fibrin gel into the electrical field. Ingeneral, when producing fibers using electroprocessing techniques,especially electrospinning, it is preferable to use the monomer of thepolymer fiber to be formed. In some embodiments it is desirable to usemonomers to produce finer filaments. In other embodiment, it isdesirable to include partial fibers to add material strength to thematrix and to provide additional sites for incorporating substances.Matrix materials such as collagen in a gelatin form may be used toimprove the ability of the material to dissolve. Acid extraction methodcan be used in preparing such gels to maintain the structure of themonomeric subunits. Units can then be treated with enzymes to alter thestructure of the monomers.

In embodiments in which two materials combine to form a third material,the solutions containing these components can be mixed togetherimmediately before they are streamed from an orifice in theelectroprocessing procedure. In this way, the third material formsliterally as the microfibers or microdroplets are formed in theelectrospinning process. Alternatively, such matrices can be formed byelectrospraying a molecule that can form matrix materials into a moistor otherwise controlled atmosphere of other molecules necessary to allowformation of the matrix to form filaments within the electric field. Forexample, fibrinogen can be sprayed into a moist atmosphere of thrombin.Materials such as fibrinogen that are capable of forming other materialssuch as fibrin can also be electrosprayed onto a target that hasthrombin. Alternatively thrombin can also be electrosprayed onto atarget that has fibrinogen.

In embodiments in which two or more matrix materials are combined toform a third (for example, combining fibrinogen and thrombin to formfibrin) the matrix materials can be electroprocessed in conjunction withor separately from each other, typically under conditions that do notallow the two molecules to form the third until the desired time. Thiscan be accomplished several ways. Using fibrinogen and thrombin as anexample, the two matrix materials can be electroprocessed from a solventthat does not allow thrombin to function. Alternatively, the fibrinogenor thrombin can be packaged in a carrier material. In this applicationthe fibrinogen is electroprocessed onto the target from one solutionsource (by itself or with a carrier), and the thrombin is deposited inan electroaerosol manner from a separate source. The thrombin can beencapsulated and sprayed as a fine mist of particles. Alternatively,thrombin and fibrinogen can be mixed with a carrier, such as PEG, orother synthetic or natural polymers such as collagen. The carrier actsto hold the reactants in place until they are initiated. These methodsare not limited to thrombin and fibrinogen and also are used withembodiments involving other combinations of matrix materials thatcombine to form a third material. The entire product is preferablystored under dry conditions to prevent the reaction of the twomaterials. When the material is placed in a moist environment, thematerials are able to combine and the product matrix material is formed.

As stated above, it is to be understood that carriers can be used inconjunction with matrix materials. Different materials, such asextracellular matrix proteins, and or substances, can be mixed with PEGor other known carriers that form filaments. For example, fibrinogen andcollagen can be mixed with PEG or other known carriers that formfilaments. This produces “hairy filaments” with the hair being fibrin.The “hairs” cross-link the surrounding matrix carrier into a gel, orprovide reactive sites for cells to interact with the substance withinthe matrix carrier, such as immunoglobulins. This approach can be usedfor forming a matrix or gelling molecules that do not normally gel. Forexample, in embodiments in which a specific type of matrix material willnot form filaments, then the matrix material can be combined with fibrinand PEG and electrosprayed to form an electroprocessed fibrin-containingmatrix. Once fibrin formation begins, a gel of the matrix material andfibrin together is produced.

Alternatively, the material can be sputtered with another molecule thatforms a sheet. Examples of molecules that form sheets include PGA, PLA,a copolymer of PGA and PLA, collagen, and fibronectin. In someembodiments, a sheet is formed with two or more materials that cancombine to form a third material. This sheet can be placed in a wetenvironment to allow conversion to the third material.

In addition to the multiple equipment variations and modifications thatcan be made to obtain desired results, similarly the electroprocessedsolution can be varied to obtain different results. For instance, anysolvent or liquid in which the material is dissolved, suspended, orotherwise combined without deleterious effect on the process or the safeuse of the matrix can be used. Materials or the compounds that formmaterials can be mixed with other molecules, monomers or polymers toobtained desired results. In some embodiments, polymers are added tomodify the viscosity of the solution. In still a further variation, whenmultiple reservoirs are used, the ingredients in those reservoirs areelectrosprayed separately or joined at the nozzle so that theingredients in the various reservoirs can react with each othersimultaneously with the streaming of the solution into the electricfield. Also, when multiple reservoirs are used, the differentingredients in different reservoirs can be phased in temporally duringthe processing period. These ingredients may include substances.

Embodiments involving alterations to the electroprocessed materialsthemselves are within the scope of the present invention. Some materialscan be directly altered, for example, by altering their carbohydrateprofile. Also, other materials can be attached to the matrix materialsbefore, during or after electroprocessing using known techniques such aschemical cross-linking or through specific binding interactions (e.g.PDGF binds to collagen at a specific binding site). Further, thetemperature and other physical properties of the process can be modifiedto obtain different results. The matrix may be compressed or stretchedto produce novel material properties.

Still further chemical variations are possible. Fibrin, for example, isformed in different ways. Building an electroprocessed matrix comprisedof fibrin, therefore, involves different ways of bringing the moleculescapable of forming fibrin, such as fibrinogen and thrombin, togetherthrough electroprocessing methods. Electroprocessed materials andmatrices can also be manipulated after they are formed with theelectroprocessing methods.

A matrix of electroprocessed fibers, in accordance with the presentinvention, can be produced as described below. In the case ofelectrospun fibrin, while any molecules capable of forming fibrin can beused, it is preferable to electroprocess fibrinogen or thrombin to makefibrin fibers.

Electroprocessing using multiple jets of different polymer solutionsand/or the same solutions with different types and amounts of substances(e.g., growth factors) can be used to prepare libraries of biomaterialsfor rapid screening. Such libraries are desired by those in thepharmaceutical, advanced materials and catalyst industries usingcombinatorial synthesis techniques for the rapid preparation of largenumbers (e.g., libraries) of compounds that can be screened. Forexample, the minimum amount of growth factor to be released and theoptimal release rate from a fibrous polymer scaffold to promote thedifferentiation of a certain type of cell can be investigated using thecompositions and methods of the present invention. Other variablesinclude fiber diameter and fiber composition. Electroprocessing permitsaccess to an array of samples on which cells can be cultured in paralleland studied to determine selected compositions which serve as promisingcell growth substrates.

Various effective conditions can be used to electroprocess a matrix.While the following is a description of a preferred method, otherprotocols can be followed to achieve the same result. Referring to FIG.1 in electrospinning fibers, micropipettes 10 are filled with materialsand suspended above a grounded target 11, for instance, a metal groundscreen placed inside the central cylinder of the RCCS bioreactor.Although this embodiment involves two micropipettes acting as sources ofmaterials, the present invention includes embodiments involving only onesource or more than two sources. A fine wire 12 is placed in thesolution to charge the solution in each pipette tip 13 to a highvoltage. At a specific voltage determined for each solution andapparatus arrangement, the solution suspended in each pipette tip isdirected towards the grounded target. This stream 14 of materials mayform a continuous filament, for example when collagen is the material,that upon reaching the grounded target, collects and dries to form athree-dimensional, ultra thin, interconnected matrix of electroprocessedcollagen fibers. Depending upon reaction conditions a single continuousfilament may be formed and deposited in a non-woven matrix.

As noted above, combinations of electroprocessing techniques andsubstances are used in some embodiments. Referring now to FIG. 2,micropipette tips 13 are each connected to micropippettes 10 thatcontain different materials or substances. The micropipettes aresuspended above a grounded target 11. Again, fine wires 12 are used tocharge the solutions. One micropipette produces a stream of collagenfibers 14. Another micropipette produces a steam of electrospun PLAfibers 16. A third micropipette produces an electroaerosol of cells 17.A fourth micropipette produces an electrospray of PLA droplets 18.Although the micropipettes are attached to the same voltage supply 15,PLA is electrosprayed rather than electrospun from the fourthmicropipette due to variation in the concentration of PLA in thesolutions. Alternatively, separate voltage supplies (not shown) can beattached to each micropipette to allow varying electroprocessing methodsto be used through application of different voltage potentials.

Similarly, referring now to FIG. 8, the same material can be applied aselectrospun fibers 19 from one of the two micropipettes andelectrosprayed droplets 20 from the other micropipette disposed at adifferent angles with respect to the grounded substrate 11. Again, themicropipette tips 13 are attached to micropipettes 10 that containvarying concentrations of materials and thus produce different types ofelectroprocessed streams despite using the same voltage supply 15through fine wires 12.

Minimal electrical current is involved in this process, and, therefore,electroprocessing, in this case electrospinning, does not denature thematerials that form the electroprocessed materials, because the currentcauses little or no temperature increase in the solutions during theprocedure. In melt electrospinning, there is some temperature increaseassociated with the melting of the material. In such embodiments, careis exercised to assure that the materials or substances are not exposedto temperatures that will denature or otherwise damage or injure them.

An electroaerosoling process can be used to produce a dense, matte-likematrix of electroprocessed droplets of material. The electroaerosolingprocess is a modification of the electrospinning process in that theelectroaerosol process utilizes a lower concentration of matrixmaterials or molecules that form electroprocessed materials during theprocedure. Instead of producing a splay of fibers or a single filamentat the charge tip of the nozzle, small droplets are formed. Thesedroplets then travel from the charged tip to the grounded substrate toform a sponge-like matrix composed of fused droplets. In someembodiments, the droplets are less than 10 microns in diameter. In otherembodiments a construct composed of fibrils with droplets, like “beadson a string” may be produced. Droplets may range in size from 100nanometers to 10 microns depending on the polymer and solvents.

As with the electrospinning process described earlier, theelectroaerosol process can be carried out using various effectiveconditions. The same apparatus that is used in the electrospinningprocess, for instance as shown in FIG. 1, is utilized in theelectroaerosol process. The differences from electrospinning include theconcentration of the materials or substances that form matrix materialsplaced in solution in the micropipette reservoir and/or the voltage usedto create the stream of droplets.

One of ordinary skill in the art recognizes that changes in theconcentration of materials or substances in the solutions requiresmodification of the specific voltages to obtain the formation andstreaming of droplets from the tip of a pipette.

The electroprocessing process can be manipulated to meet the specificrequirements for any given application of the electroprocessedcompositions made with these methods. In one embodiment, themicropipettes can be mounted on a frame that moves in the x, y and zplanes with respect to the grounded substrate. The micropipettes can bemounted around a grounded substrate, for instance a tubular mandrel. Inthis way, the materials or molecules that form materials streamed fromthe micropipettes can be specifically aimed or patterned. Although themicropipettes can be moved manually, the frame onto which themicropipettes are mounted is preferably controlled by a microprocessorand a motor that allow the pattern of streaming collagen to bepredetermined by a person making a specific matrix. Such microprocessorsand motors are known to one of ordinary skill in the art. For instance,matrix fibers or droplets can be oriented in a specific direction, theycan be layered, or they can be programmed to be completely random andnot oriented.

In the electrospinning process, the stream or streams can branch out toform fibers. The degree of branching can be varied by many factorsincluding, but not limited to, voltage, ground geometry, distance frommicropipette tip to the substrate, diameter of micropipette tip, andconcentration of materials or compounds that will form theelectroprocessed materials. As noted, not all reaction conditions andpolymers may produce a true multifilament, under some conditions asingle continuous filament is produced. Materials and variouscombinations can also be delivered to the electric field of the systemby injecting the materials into the field from a device that will causethem to aerosol. This process can be varied by many factors including,but not limited to, voltage (for example ranging from about 0 to 30,000volts), distance from micropipette tip to the substrate (for examplefrom 0-40 cm), the relative position of the micropipette tip and target(i.e. above, below, aside etc.), and the diameter of micropipette tip(approximately 0-2 mm). Several of these variables are well-known tothose of skill in the art of electrospinning microfiber textile fabrics.

The geometry of the grounded target can be modified to produce a desiredmatrix. By varying the ground geometry, for instance having a planar orlinear or multiple points ground, the direction of the streamingmaterials can be varied and customized to a particular application. Forinstance, a grounded target comprising a series of parallel lines can beused to orient electrospun materials in a specific direction. Thegrounded target can be a cylindrical mandrel whereby a tubular matrix isformed. Most preferably, the ground is a variable surface that can becontrolled by a microprocessor that dictates a specific ground geometrythat is programmed into it. Alternatively, for instance, the ground canbe mounted on a frame that moves in the x, y, and z planes with respectto a stationary micropipette tip streaming collagen.

The substrate onto which the materials are streamed, sprayed orsputtered can be the grounded target itself or it can be placed betweenthe micropipette tip and the grounded target. The substrate can bespecifically shaped, for instance in the shape of a nerve guide, skinpatch, fascial sheath, or a vascular graft for subsequent use in vivo.The electroprocessed compositions can be shaped to fit a defect or siteto be filled. Examples include a site from which a tumor has beenremoved, an injury site in the skin (a cut, a biopsy site, a hole orother defect) and a missing or shattered piece of bone. Theelectroprocessed compositions may be shaped into shapes useful forsubstance delivery, for example, a skin patch, a lozenge for ingestion,an intraperitoneal implant, a subdermal implant, the interior lining ofa stent, a cardiovascular valve, a tendon, a ligament a dentalprosthesis, a muscle implant, or a nerve guide. Electroprocessing allowsgreat flexibility and allows for customizing the construct to virtuallyany shape needed. Many matrices are sufficiently flexible to allow themto be formed to virtually any shape. In shaping matrices, portions ofthe matrix may be sealed to one another by, for example, heat sealing,chemical sealing, and application of mechanical pressure or acombination thereof. An example of heat sealing is the use ofcrosslinking techniques discussed herein to form crosslinking betweentwo portions of the matrix. Sealing may also be used to close an openingin a shaped matrix. Suturing may also be used to attach portions ofmatrices to one another or to close an opening in a matrix. It has beenobserved that inclusion of synthetic polymers enhances the ability ofmatrices to be heat sealed.

Other variations of electroprocessing, particularly electrospinning andelectroaerosoling include, but are not limited to the following:

1. Using different solutions to produce two or more different fibers ordroplets simultaneously (fiber or droplet array). In this case, thesingle component solutions can be maintained in separate reservoirs.

2. Using mixed solutions (for example, materials along with substancessuch as cells, growth factors, or both) in the same reservoir(s) toproduce fibers or droplets composed of electroprocessed materials aswell as one or more substances (fiber composition “blends”).Nonbiological but biologically compatible material can be mixed with abiological molecule.

3. Utilizing multiple potentials applied for the different solutions orthe same solutions.

4. Providing two or more geometrically different grounded targets (i.e.small and large mesh screens).

5. Placing the mold or mandrel or other ungrounded target in front ofthe grounded target.

6. Applying agents such as Teflon onto the target to facilitate theremoval of electroprocessed materials from the target (i.e. make thematerial more slippery so that the electroprocessed materials do notstick to the target).

7. Forming an electroprocessed material that includes materials appliedusing multiple electroprocessing methods. For example, electrospunfibers and electroaerosol droplets in the same composition can bebeneficial for some applications depending on the particular structuredesired. This combination of fibers and droplets can be obtained byusing the same micropipette and solution and varying the electricalcharge; varying the distance from the grounded substrate; varying thepolymer concentration in the reservoir, using multiple micropipettes,some for streaming fibers and others for streaming droplets; or anyother variations to the method envisioned by those of skill in this art.The fibers and droplets can be layered or mixed together in same layers.In applications involving multiple micropipettes, the micropipettes canbe disposed in the same or different directions and distances withreference to the target.

8. Using multiple targets.

All these variations can be done separately or in combination to producea wide variety of electroprocessed materials and substances.

The various properties of the electroprocessed materials can be adjustedin accordance with the needs and specifications of the cells to besuspended and grown within them. The porosity, for instance, can bevaried in accordance with the method of making the electroprocessedmaterials or matrix. Electroprocessing a particular matrix, forinstance, can be varied by fiber (droplet) size and density. If thecells to be grown in the matrix require a great deal of nutrient flowand waste expulsion, then a loose matrix can be created. On the otherhand, if the tissue to be made requires a very dense environment, then adense matrix can be designed. Porosity can be manipulated by mixingsalts or other extractable agents. Removing the salt will leave holes ofdefined sizes in the matrix.

One embodiment for appropriate conditions for electroprocessing fibrinis presented below. For electroprocessing fibrin by combining fibrinogenand thrombin, the appropriate approximate ranges are: voltage 0-30,000volts; pH 7.0 to 7.4; calcium 3 to 10 mM; temperature 20 to 40° C.;ionic strength 0.12 to 0.20 M; thrombin 0.1 to 1.0 units per ml; andfibrinogen 5 to 25 mg/ml. For electroprocessing fibrin monomer, the pHstarts at 5 and increases to 7.4 while the ionic strength starts above0.3 M and decreases to 0.1 M. The other conditions are similar as statedwithin this paragraph. Electroprocessed fibrin matrices of varyingproperties can be engineered by shifting the pH, changing the ionicstrength, altering the calcium concentration, or adding additionalpolymeric substrates or cationic materials. For electroprocessingcollagen, the appropriate approximate ranges are: voltage 0-30,000volts; pH 7.0 to 8.0; temperature 20 to 42° C.; and collagen 0 to 5mg/ml. Electroprocessed collagen matrices of varying properties can beengineered by shifting the pH, changing the ionic strength (e.g.addition of organic salts), or adding additional polymeric substrates orcationic materials.

Shapes of Electroprocessed Materials and Matrices

Electroprocessed materials can be electrodeposited inside a specificallyshaped mold. For instance, a particular type of organ or tissue that tobe replaced has a specific shape, such as a skin patch to fit a biopsysite or a large scalp area following a wide area removed afterdiscovering a malignant melanoma. That shape is then reproduced andcreated inside a mold designed to mimic that shape. This mold can befilled by electrodepositing the material into it. In this way, thematrix exactly mimics the mold shape. In some embodiments, matrices thatwill become extracellular matrices and that have a specific shape areused in the creation of a new organ. Hollow and solid organs can bemade. Mixing cells with the material during electrospraying forms cellswithin the matrix so that they do not have to migrate into a matrix.

Methods of Combining Substances with Electroprocessed Materials

Substances can be combined with the electroprocessed materials by avariety of means. In some embodiments, the substance comprises moleculesto be released from the electroprocessed material and is therefore addedto or incorporated within the matrix of electroprocessed material.Substances can be mixed in the solvent carriers or solutions ofmaterials for electroprocessing. In this system materials can be mixedwith various substances and directly electroprocessed. The resultingcomposition comprising an electroprocessed matrix and substance can betopically applied to a specific site and the substances released fromthe material as a function of the material undergoing breakdown in thesurrounding environment. Substances may also be released from theelectroprocessed compositions of the present invention throughdiffusion.

The state of the electroprocessed material in relation to theincorporated substances is dictated and can be controlled by thechemistry of the system and varies based on the selection of matrixmaterials, solvent(s) used, and solubility of the matrix materials inthose solvents. These parameters can be manipulated to control therelease of the substances (or other elements into the surroundingenvironment). If substances to be incorporated into the electroprocessedmaterial are not miscible with the material, separate solvent reservoirsfor the different components can be used. Mixing in such an embodimentoccurs prior to, during, and/or after deposition on the target, or acombination thereof. It is to be understood that substances may beentrapped or entangled within an electroprocessed material, bonded to amaterial before the material undergoes electroprocessing, or bound tospecific sites within the matrix material.

In a variation of this embodiment, the substance is a particle oraggregate comprising a matrix of compounds or polymers such as alginatethat, in turn, contain one or more compounds that will be released fromthe electroprocessed material. Drugs can be combined with alginate by,for example, combining a drug suspension or drug particulate in thealginate in the presence of calcium. Alginate is a carbohydrate thatforms aggregates when exposed to calcium. the aggregates can be used totrap drugs. The aggregates dissolve over time, releasing the trappedsubstances, such as cells trapped in alginate. The particles, which arethen incorporated within the larger electroprocessed matrix, arebiologically compatible but relatively stable and will degradegradually. In some circumstances, the electroprocessed materialsresemble a string of pearls. This is a physical aspect of theelectroprocessing. If the polymer concentration is low, electrosprayingof beads occurs. As polymer concentration increases there are some beadsand some fibers. A further increase in polymer concentration leads topredominantly or all fibers. Therefore, the appearance of the pearls ona string is a transition phase.

If a drug (for example, penicillin) does not bind or interact with anelectrospun matrix material, the drug can be entrapped in PGA or PLApellets or electroaerosoled to produce pellets in the electrospunmaterial. The pellets or electroaerosoled droplets containing the drugbegin to dissolve after administration to deliver the entrappedmaterial. Some agents can be coupled to synthetic, or natural polymer bya covalent bond, prior to or after spinning.

In other embodiments, the substance is electroprocessed. Substances canbe electroprocessed from the same orifice as the materials or fromdifferent orifices. Substances can also be subjected to the same or adifferent type of electroprocessing as the material. A molecule can bebonded to the electroprocessed material directly or through linking to amolecule that has an affinity for the material. An example of thisembodiment involves bonding polypeptide substances to heparin, which hasan affinity for collagen materials. This embodiment allows releaserelate to be controlled by controlling the rate of degradation of thematerial, for example by enzymatic or hydrolytic breakdown.

In other embodiments, the electroprocessed material can entrap substanceduring the electrodeposition process. This can be accomplished bydisposing substances in the space between the source of theelectroprocessed stream and the target for the electroprocessedmaterial. Placing such substances in the space between the source andtarget can be accomplished by a number of methods, including but notlimited to, suspending in air or other gases, dripping, spraying, orelectroprocessing the substances. The substances can be present in thatspace in, for example, particulate, aerosol, colloidal, or vapor form.In these embodiments, the electroprocessed material or matrix willphysically entrap the substances. This embodiment can also be used toencapsulate larger particles, such as cells, large particles, ortablets. For example, if a tablet is dropped through the matrix as itforms, the tablet is surrounded by the matrix. If a small object, like acell is dropped through the matrix as it forms or placed in an aerosolwithin the matrix, the object may be trapped between filaments, withinfilaments or “attached to the outside of the filaments. For example, bysuspending cells in a solution or within a matrix, the cells can becomepart of an electrospun matrix during fabrication of the filaments.Alternatively, encapsulation can occur by dropping substances onto orthrough a matrix material stream as a matrix forms. The cells thusbecome surrounded by a matrix of electroprocessed material. Theseembodiments can be used to incorporate within a matrix substances thatare not soluble and/or are too large to form a suspension in the solventused for the production of the material. For substances in a mist orvapor form, controlling distribution and composition of substances inthe space between the source and target can be used to alter thephysical and chemical properties of the electroprocessed material andthe pattern of distribution of the substances in the electroprocessedmaterial. For all of the foregoing embodiments, the substances can beplaced in the electroprocessed material in capsules, vesicles, or othercontainments for subsequent release. Since the solvent carrier oftenevaporates in the electroprocessing technique as the electroprocessedmaterial forms, such as a filament, substances may be placed in theelectroprocessed matrix and solvent toxicity is greatly reduced oreliminated.

In embodiments wherein the substance comprises cells, the cells can, forexample, be suspended in a solution or other liquid that contains thematerial to be electroprocessed, disposed in the area between thesolutions and target, or delivered to a target or substrate from aseparate source before, during, or after electroprocessing. Cells can bedripped through the matrix, onto the matrix as it deposits on the targetor suspended within an aerosol as a delivery system for the cells to theelectroprocessed material. The cells can be delivered in this mannerwhile the matrix is being formed. As an example, cardiac fibroblastswere suspended in phosphate-buffered saline (PBS) at a concentration ofapproximately one million cells per milliliter. The suspension of cellswas placed within a reservoir of a Paasche air brush. To test theefficacy of using this type of device to deliver cells, the cellsuspension was initially sprayed onto a 100 mm culture dish. Some of thecells survived, attached to the dish and spread out over the substratum.In a second trial, the culture dish was located further away from theair brush and the experiment was repeated. Cells were observed on thedish. They appeared to be flattened by the impact and were partiallyspread out over the surface of the substratum. Culture media was addedto the dish and the cells were placed into an incubator. After one hourof culture, the cells were inspected again, and many were found to havespread out further over the substratum. These results demonstrate that asimple airbrush device can be used to place cells into an aerosoldroplet and deliver them on demand to a surface or site of interest.Cell viability can be improved by restricting this technique to cellsthat are resistant to the shear forces produced in the technique,developing a cell suspension with additives that cushions the cells orrefining the aerosolizing device to produce a more laminar flow. Inaddition, directing the cell aerosol into matrix materials as the matrixis forming in the space between the target or mandrel and the source(s)of molecules being electroprocessed produces the effect of cushioningthe cells. While not wanting to be bound by the following statement, itis believed that the cells will be trapped in the storm of filaments orother bodies produced by electrospinning or electroprocessing and pulledonto the mandrel. This situation may be less traumatic to the cells thandirectly spraying the cells onto a solid surface.

In one embodiment, the cells are added either before or at the same timeas the materials or compounds that form electroprocessed materials arebrought together. In this way, the cells are suspended throughout thethree-dimensional matrix. In embodiments in which the electroprocessedmaterial comprises fibrin formed by combining thrombin and fibrinogen,the cells are typically included in the mixture that contains thefibrinogen (whether it is plasma or purified fibrinogen). Whenevermaterials comprise two or more separate materials that combine to form adifferent material (such as fibrinogen and thrombin) bringing thematerials together immediately prior to insertion into a mold, orimmediately prior to the streaming step in the electrospinning processhelps result in a good distribution of cells in suspension in theresulting extracellular matrix.

Cells can be added as the filaments are produced in the space betweenthe target and polymer source. This is accomplished by dripping thecells onto the target, dripping the cells into the matrix as it forms,aerosoling the cells into the matrix or onto the target orelectrospraying the cells into the matrix as it condenses and forms nearor on the grounded target. In another embodiment, cells are sprayed ordribbled into a forming electroprocessed material or matrix and therebytrapped as the electroprocessed material crosses the air gap between thesource solutions and target.

An alternative method to deliver cells to an electroprocessed materialinvolves electroaerosol delivery of the cells. Cells can be deposited byelectrostatic spraying at, for example, 8 kV directly onto standardpolystyrene culture dishes, suggesting that electrostatic cell sprayingis a viable approach. Cardiac fibroblasts in phosphate buffered saline(PBS) have been electroaerosoled up to a 20 Kv potential difference. Inanother example, Schwann cells (rat) were plated on a PS petri dish byconventional methods after one day. Schwann cells were alsoelectrosprayed onto a PS petri dish with a metal ground plate behind thedish at 10 kV after one day. Both samples grew to almost confluenceafter one week. The electroaerosol approach provides some distinctadvantages. First, the shear forces produced during the delivery phase(i.e. the production of the aerosol) appear to be much less traumatic tothe cells. Second, the direction of the aerosol can be controlled with ahigh degree of fidelity. In essence the cell aerosol can be painted ontothe surface of interest. This allows the cell to be targeted to specificsites. In electroaerosol delivery, cells are suspended in an appropriatemedia (e.g. culture media, physiological salts, etc.) and charged to avoltage, and directed towards a grounded target. This process is verysimilar to that used in electroprocessing, particularly electrospinning.The produces a fine mist of cells trapped within the droplets as theyare produced and directed at the grounded target.

Cells can be delivered using aerosol and electroaerosol techniques ontoan electroprocessed material that is forming by an electroprocessingtechnique. The electroaerosol of cells can be delivered in parallel(i.e. alongside) the electroprocessing material or from a separate site.The cells can be delivered to the storm of filaments or particlesproduced within the air gap in the electrodeposition process or directedat the target. The cells and electroprocessed material also can bedelivered in an alternating sequence to the target, i.e. electrodepositthe material, aerosol the cells, electrodeposit the material, aerosolthe cells. This allows for the discrete layering of the construct inseparate layers. Furthermore, a vapor source can be provided thatdirects water onto the mandrel of target used to collect the cells.Providing this moisture improves cell viability by keeping the cellsfrom dehydrating during processing. Cells can be added to theelectroprocessed material at any time or from any orientation in anyaerosol strategy. Again the advantage of the process in general is thatcollagen, for example, collects in a dried state on the target mandrel.Accordingly, although some solvents for collagen may be toxic, they arelost from the system before the filaments collect on the target.

Cells can also be trapped within a carrier prior to producing anaerosol. For example, cells can be encapsulated within a material likealginate. The encapsulated cells are physically protected from shear andtrauma during processing. Cells delivered in this form to theelectroprocessed material will have higher viability when sprayed orelectrostatically seeded.

An electroaerosol or otherwise electroprocessed material can also bedelivered directly to an in situ site. For example, an electroprocessedmaterial can be produced directly onto a skin wound, with or withoutsubstances such as molecules or cells. Additional cells or materials canthen be aerosolized onto or into the wound site. Other surgical sitescan also be amenable the delivery of materials using variouselectrodeposition techniques or combinations thereof of these methods.

In other embodiments, substances can be applied to the electroprocessedmaterial after formation, for example by soaking the electroprocessedmaterial in the substance or by spraying the substance onto theelectroprocessed material.

Persons skilled in the art will recognize that more than one method forcombining the substances with electroprocessed materials can be used ina single embodiment or application. Combining methods can be especiallyuseful in embodiments involving release of more than one compound orcompounds intended to have complex release kinetics, although suchcombinations are not limited to those embodiments.

Magnetically and electrically active materials can be electroprocessed,including, for example, preparing conducting polymer fibers produced byelectrospinning. In addition, conducting polymers can be preparedin-situ in the matrix by, for example, incorporation of a monomer (e.g.,pyrrole) followed by treatment with polymerization initiator and oxidant(e.g., FeCl₃). Finally, conducting polymers can be grown in the materialafter electroprocessing by using a matrix-coated conductor as the anodefor electrochemical synthesis of, for example, polypyrrole orpolyaniline. Compounds that can form electroprocessed materials can beadded to an aqueous solution of pyrrole or aniline to create aconducting polymer at the anode with the entrapped electroprocessedmaterial-forming compounds, which can then be treated with othercompounds to allow formation of the material to occur.

Patterns of Electroprocessed Materials and Substance Distribution

Many embodiments of the present invention involve means for manipulatingthe pattern or distribution of electroprocessed materials and/orsubstances within an electroprocessed material. For example, anelectroprocessing target can also be specifically charged or groundedalong a preselected pattern so that electroprocessed materials streamedtoward the target are directed into specific directions or distributionson the target or on a substrate. The electric field can be controlled bya microprocessor to create a matrix having a desired geometry. Thetarget and the electroprocessing nozzle or nozzles can be movable withrespect to each other and to the target thereby allowing additionalcontrol over the geometry of the electroprocessed material to be formed.In embodiments in which substances are electroprocessed, thismanipulation will also allow control of the distribution of substanceswithin the electroprocessed materials. For example a matrix can beprepared on a mandrel, and substances from a separate reservoir can besprayed, dripped, electroprocessed in a specific pattern over theexisting matrix. This may also be accomplished by simultaneouslyelectrospraying a matrix from one source and a substance from anothersource. In this example the matrix source may be stationary and thesubstance source is moved with respect to the target mandrel.

Other features that allow establishment of such a pattern include, butare not limited to, the ability to deposit multiple layers of the sameor different materials, combining different electroprocessing methods,the use multiple orifices with different contents for electroprocessing,and the existence of numerous methods for combining substances with thematerials. For example, a gradient of substances can be created along aelectroprocessed material. In embodiments in which the matrix is shapedinto a cylindrical construct, for example, the gradient can be preparedalong the long axis of a construct (left to right) or the perpendicularaxis (inside to out). This configuration is used to provide achemoattractant gradient to guide the movement of cells within aspecified site. Thus, for example, in some embodiments in whichneovascular agents are prepared in a perpendicular gradient along acollagen-based construct, the agents can be concentrated on the dorsalsurface of a sheet of the material. The ventral side can be placedagainst a wound and the higher concentration of angiogenic materials onthe dorsal surface of the construct will increase the migration ofendothelial cells through the electrospun material. Again, embodimentswith complex patterns can use a microprocessor programmed with thespecific parameters to obtain a specific, preselected electroprocessedpattern of one or more electroprocessed polymers, optionally with one ormore substances.

Uses for the Compositions of the Present Invention

Substance Delivery

One use of the compositions of the present invention is the delivery ofone or more substances to a desired location. In some embodiments, theelectroprocessed materials are used simply to deliver the materialsitself. In other embodiments, the electroprocessed materials are used todeliver substances that are contained in the electroprocessed materialor that are produced or released by substances contained in theelectroprocessed material. For example, an electroprocessed materialcontaining cells can be implanted in a body and used to delivermolecules produced by the cells after implantation. The presentcompositions can be used to deliver substances to an in vivo location,an in vitro location, or other locations. The present compositions canbe administered to these locations using any method.

In the field of substance delivery, the compositions of the presentinvention have many attributes that allow delivery of substances using awide variety of release profiles and release kinetics. For example,selection of the substance and the method by which the substance iscombined with the electroprocessed material affects the substancerelease profile. To the extent that the substances are not immobilizedby the electroprocessed material, release from the electroprocessedmaterial is a function of diffusion. An example of such an embodiment isone in which the substance is sprayed onto the electroprocessedmaterial. To the extent that the substances are immobilized by theelectroprocessed material, release rate is more closely related to therate at which the electroprocessed material degrades. An example of suchan embodiment is one in which the substance is covalently bonded to theelectroprocessed material. For a substance is trapped within anelectrospun aggregate or filament, release kinetics would be determinedby the rate at which the surrounding material degrades or disintegrates.Still other examples are substances that are coupled to theelectroprocessed material by a light sensitive bond. Exposing such abond to light releases the substance from the electroprocessed material.Conversely, in some embodiments of this invention, materials can beexposed to light to cause binding of agents in vivo or in vitro.Combining the compound with the electroprocessed material in solution,rather than in suspension, will result in a different pattern of releaseand thereby provide yet another level of control for the process.Further, the porosity of the electroprocessed material can be regulated,which affects the rate of release of a substance. Enhanced porosityfacilitates release. Substance release is also enhanced by fragmentingor pulverizing the electroprocessed material. Pulverized material can,for example be applied to a wound site, ingested or formed into anothershape such as a capsule or a tablet. In embodiments in which thesubstance is present in the form of a large particle such as a tabletencapsulated in the electroprocessed material or a molecule trappedinside an electroprocessed filament, release is dictated by a complexinterplay of the rate the particles dissolve or degrade and anybreakdown or degradation of the electroprocessed material structure. Inembodiments in which the substance comprises cells that will express oneor more desired compounds, factors that affect the function andviability of the cells and the timing, intensity, and duration ofexpression can all affect the release kinetics. Chemicals that affectcell function, such as oligonucleotides, promoters or inhibitors of celladhesion, hormones, and growth factors, for example, can be incorporatedinto the electroprocessed material and the release of those substancesfrom the electroprocessed material can provide a means of controllingexpression or other functions of cells in the electroprocessed material.

Release kinetics in some embodiments are manipulated by cross-linkingelectroprocessed material through any means. In some embodiments,cross-linking will alter, for example, the rate at which theelectroprocessed material degrades or the rate at which a compound isreleased from the electroprocessed material by increasing structuralrigidity and delaying subsequent dissolution of the electroprocessedmaterial. Electroprocessed materials can be formed in the presence ofcross-linking agents or can be treated with cross-linking agents afterelectrodeposition. Any technique for cross-linking materials may be usedas known to one of ordinary skill in the art Examples of techniquesinclude application of cross-linking agents and application of certaincross-linking radiations. Examples of cross-linking agents that workwith one or more proteins include but are not limited to condensingagents such as aldehydes e.g., glutaraldehyde, carbodiimide EDC(1-ethyl-3(3 dimethyl aminopropyl)), photosensitive materials that crosslink upon exposure to specific wavelengths of light, osmium tetroxide,carbodiimide hydrochloride, and NHS (n-hydroxysuccinimide), and FactorXIIIa. Ultraviolet radiation is one example of radiation used tocrosslink matrix materials in some embodiments. Natural materials can becross-linked with other natural materials. For example, collagen can becross-linked and or stabilized by the addition of fibronectin and orheparin sulfate. For some polymers heat can be used to alter the matrixand cross link elements of the matrix by fusing adjacent components ofthe construct. Polymers may also be partially solubilized to alter thestructure of the material, for example brief exposure of some syntheticsto alcohols or bases can partially dissolve and anneal adjacentfilaments together. Some polymers may be cross-linked using chemicalfusion or heat fusion techniques. Synthetic polymers generally can becross-linked using high energy radiation (e.g., electron beams, gammarays). These typically work by the creation of free radicals on thepolymer backbone which then couple, affording cross links. Backbone freeradicals can also be generated via peroxides, azo compounds, arylketones and other radical-producing compounds in the presence of heat orlight. Reduction-oxidation reactions that produce radicals (e.g.,peroxides in the presence of transition metal salts) can also be used.In many cases, functional groups on polymer backbones or side chains canbe reacted to form cross-links. For example, polysaccharides can betreated with diacylchlorides to form diester cross-links. Cross-linkingmay also occur after application of a matrix where desirable. Forexample, a matrix applied to a wound may be cross-linked afterapplication to enhance adhesion of the matrix to the wound.

The release kinetics of the substance is also controlled by manipulatingthe physical and chemical composition of the electroprocessed material.For example, small fibers of PGA are more susceptible to hydrolysis thanlarger diameter fibers of PGA. An agent delivered within anelectroprocessed material composed of smaller PGA fibers is releasedmore quickly than when prepared within a material composed of largerdiameter PGA fibers.

In some embodiments substances such as peptides can be released in acontrolled manner in a localized domain. Examples include embodiments inwhich the substance is chemically or covalently bonded to theelectroprocessed material. The formation of peptide gradients is acritical regulatory component of many biological processes, for examplein neovasculogenesis. In surgical applications, anti-vascular peptidesor anti-sense oligonucleotides can be incorporated into anelectroprocessed material that is then wrapped around or placed within atumor that is inaccessible to conventional treatments to allow forlocalized release and effect. Release of the anti-vascular substancessuppresses tumor growth. Antisense oligonucleotides can be released fromthe construct into the tumor and used to suppress the expression genesequences of interest. In another example anti-sense sequences directedagainst gene sequences that control proliferation can be deliveredwithin an electroprocessed matrix coated stent. The stretch normallyassociated with the placement of the stent initiates smooth muscle cellproliferation, and anti-sense sequences designed to suppress celldivision reduce the deleterious effects of the smooth muscle cellproliferation associated with the procedure. In another embodiments, theelectroprocessed material delivers sense and antisense oligonucleotidesto promote or to inhibit localized cell function for a period of time.For example, an antisense oligonucleotide is released from anelectroprocessed material to suppress the expression of a deleteriousenzyme in a wound. Examples of such enzymes are matrixmetalloproteinases (MMPs), which are often overexpressed in chronicwounds. In another example, the electroprocessed material applied to awound releases plasmids that contain nucleotide sequences coding fortissue inhibitors of metalloproteinases (TIMPs). Cells in the wound willexpress TIMPs, resulting in local delivery of TIMPs that will inhibitMMP function.

Physical processing of the formed electroprocessed material is anotherway to manipulate release kinetics. In some embodiments, mechanicalforces, such as compression, applied to an electroprocessed materialhasten the breakdown of the matrix by altering the crystalline structureof the material. Structure of the matrix is thus another parameter thatcan be manipulated to affect release kinetics. Polyurethanes and otherelastic materials such as poly(ethylene-co-vinyl acetate), silicones,and polydienes (e.g., polyisoprene), polycaprolactone, polyglycolic acidand related polymers are examples of materials whose release rate can bealtered by mechanical strain.

Release kinetics can also be controlled by preparing laminatescomprising layers of electroprocessed materials with differentproperties and substances. For example, layered structures composed ofalternating electroprocessed materials can be prepared by sequentiallyelectroprocessing different materials onto a target. The outer layerscan, for example, be tailored to dissolve faster or slower than respectthe inner layers. Multiple agents can be delivered by this method,optionally at different release rates. Layers can be tailored to providea complex, multi-kinetic release profile of a single agent over time.Using combinations of the foregoing can provide for release of multiplesubstances released, each with a complex profile.

Suspending a substance in particles that are incorporated in theelectroprocessed material provides another means for controlling releaseprofile. Selection of the composition of these smaller particle matricesprovides yet another way to control the release of compounds from theelectroprocessed material. The release profile can be tailored by thecomposition of the material used in the process.

Embodiments also exist in which the substances are contained inliposomes or other vesicles in the electroprocessed matrix. Vesicles areprepared that will release one or more compounds when placed in fluidsat a specific pH range, temperature range, or ionic concentration.Methods for preparing such vesicles are known to persons of skill in theart. The electroprocessed material can be delivered to a site ofinterest immediately or is stored either dry or at a pH at which releasewill not occur, and then delivered to a location containing liquids thathave a pH at which release will occur. An example of this embodiment isan electroprocessed material containing vesicles that will release adesired compound at the pH of blood or other fluids released from awound. The matrix is placed over a wound and releases fluids upondischarge of fluids from the wound.

Incorporating constituents that are magnetically sensitive orelectrically sensitive into the electroprocessed material providesanother means of controlling the release profile. A magnetic or electricfield can then be subsequently applied to some or all of the matrix toalter the shape, porosity and/or density of the electroprocessedmaterial. For example, a field can stimulate movement or conformationalchanges in the matrix due to the movement of magnetically orelectrically sensitive particles. Such movement can affect the releaseof compounds from the electroprocessed material. For example, alteringthe conformation of the material can increase or decrease the extent towhich the material is favorable for compound release.

In some embodiments, magnetic or electrically sensitive constituentsthat have been processed or co-processed with an electroprocessedmaterial can be implanted subdermally to allow delivery of a drug over along interval of time. By passing a magnetic field or an electricalfield across the material, drug release is induced. The electroprocessedmaterial structure is stable and does not substantially change withoutelectromagnetic stimulation. Such embodiments provide controlled drugdelivery over a long period of time. For example, an electroprocessedmaterial that has magnetic or electrical properties and insulin can befabricated and placed subdermally in an inconspicuous site. By passing amagnetic field or an electrical field across the composition, insulinrelease can be induced. A similar strategy may be used to releasecompounds from a construct that has light sensitive elements, exposingthese materials to light will either cause the material itself tobreakdown and or cause the release of substances that are bound to theelectroprocessed material by the light sensitive moiety.

In other embodiments, the substances comprise vesicles encapsulatedwithin the electroprocessed material along with electrical or magneticmaterials. The vesicles contain a compound to be released from thevesicles. Placing an electrical or magnetic field across theelectroprocessed material causes the compounds within the vesicles canbe released by, for example, deforming the vesicles to the point ofrupture or by changing the permeability (in some cases reversibly) ofthe vesicle wall. Examples of these embodiments include transfectionagents, such as liposomes, that contain nucleic acids that enhance theefficiency of the process of gene delivery to the cell.

In other embodiments, the composition comprising an electroprocessedmaterial and substance is used as a transdermal patch for localizeddelivery of medication, or of a component of such a patch. In some ofthese embodiments, electrically conductive materials are incorporatedinto such a composition, which is then used as a component of aniontophoresis system in which one or more substances is delivered inresponse to the passage of electric current. Electrically conductivematerials can have a direct healing effect on bone injuries. For exampleplacing a small electric current across a fracture site promoteshealing. An electroprocessed bone mimetic that conducts or producescurrent can be made and placed within a fracture. The addition of theelectrical current will promote healing at a rate that is faster thanthe addition of the electroprocessed composition alone.

In other embodiments, an electroprocessed material or a portion thereofcontaining electromagnetic properties is stimulated by exposure to amagnet to move and thereby to apply or to release physical pressure to apressure-sensitive capsule or other enclosure that contains molecules tobe released from the material. Depending on the embodiment, the movementwill affect the release relate of the encapsulated molecules.

Response of the composition to electric and magnetic fields can beregulated by features such as the composition of the electroprocessedmaterial, size of the filaments, and the amount of conductive materialadded. Electromechanical response from polyaniline is the result ofdoping-induced volume changes, whereas ion gradients leading osmoticpressure gradients are responsible for field-induced deformation inionic gels such as poly(2-acrylamido-2-methyl propanesulfonicacid). Ineach case, ion transport kinetics dominates the response, and faciletransport is observed with the small fibers. Gel swelling and shrinkingkinetics have been shown to be proportional to the square of thediameter of a gel fiber. Electromechanical response times of fiberbundles of less than 0.1 s, are possible in the regime of typicalmuscle.

Embodiments involving delivery of molecules produced by cells providemany means by which rejection and immune response to cells can beavoided. Embodiments using cells from a recipient thus avoid theproblems associated with rejection and inflammatory and immunologicalresponse to the cells. In embodiments in which cells from an organismother than the recipient are used, the matrix can sequester the cellsfrom immune surveillance by the recipient's immune system. Bycontrolling parameters such as the pore size of the electroprocessedmaterial or matrix, nutritive support to the cells trapped in the matrixcan be permitted while the cells are protected from detection andresponse by the recipient's immune system. As an example, pancreaticislet cells that manufacture insulin collected from a donor can beencapsulated in an electroprocessed matrix and implanted in a recipientwho cannot make insulin. Such an implant can be placed, for example,subdermally, within the liver, or intramuscularly. For some immuneresponses permanent sequestration from the host system may not benecessary. The electroprocessed material can be designed to shield theimplanted material for a given length of time and then begin tobreakdown. In still other embodiments, bacteria or other microbialagents engineered to manufacture the desired compound can be used. Thisembodiment provides the advantages of using cells that are more easilymanipulated than cells from the recipient or a donor. Again, theelectroprocessed material can serve to shield the bacteria from immuneresponse in this embodiment. The advantage of using a bacteria carrieris that these microbes are more easily manipulated to express a widevariety of products. Embodiments in which cells are transientlytransfected allow for expression to be limited to a defined period.Transient genetic engineering allows cells to revert to their originalstate in embodiments in which such reversion is desired to minimize therisks of complications.

In some embodiments, cells are genetically engineered such that theexpression of a specific gene may be promoted or inhibited throughvarious means known in the art. For example, a tetracycline sensitivepromoter can be engineered into a gene sequence. That sequence is notexpressed until the tetracycline is present. Cell markers or bacterialmarkers can also be used to identify the inserted material. For example,green fluorescent proteins placed within an engineered genetic materialglow green when expressed. Embodiments using this feature allowverification of the viability of the cells, bacteria, or gene sequencesin a matrix. The visibility of such a marker also assists in recoveringan implanted electroprocessed composition.

Although the present invention provides versatility in release kinetics,embodiments also exist in which one or more substances are not releasedat all from the electroprocessed material. Substances may perform afunction at a desired site. For example, in some embodiments, antibodiesfor a specific molecule are immobilized on an electroprocessed matrixand the composition is placed at a desired site. In this embodiment, theantibodies acts to bind the molecules in the vicinity of thecomposition. This embodiment is useful for isolating molecules that bindto an antibody. Another example is an electroprocessed matrix containingimmobilized substrates that will bind irreversibly to an undesirableenzyme and thereby inactivate the enzyme.

The compositions of the present invention may be combined withpharmaceutically or cosmetically acceptable carriers and administered ascompositions in vitro or in vivo. Forms of administration include butare not limited to injections, solutions, creams, gels, implants, pumps,ointments, emulsions, suspensions, microspheres, particles,microparticles, nanoparticles, liposomes, pastes, patches, tablets,transdermal delivery devices, sprays, aerosols, or other means familiarto one of ordinary skill in the art. Such pharmaceutically orcosmetically acceptable carriers are commonly known to one of ordinaryskill in the art. Pharmaceutical formulations of the present inventioncan be prepared by procedures known in the art using well known andreadily available ingredients. For example, the compounds can beformulated with common excipients, diluents, or carriers, and formedinto tablets, capsules, suspensions, powders, and the like. Examples ofexcipients, diluents, and carriers that are suitable for suchformulations include the following fillers and extenders (e.g., starch,sugars, mannitol, and silicic derivatives); binding agents (e.g.,carboxymethyl cellulose and other cellulose derivatives, alginates,gelatin, and polyvinyl-pyrrolidone); moisturizing agents (e.g.,glycerol); disintegrating agents (e.g., calcium carbonate and sodiumbicarbonate); agents for retarding dissolution (e.g., paraffin);resorption accelerators (e.g., quaternary ammonium compounds); surfaceactive agents (e.g., cetyl alcohol, glycerol monostearate); adsorptivecarriers (e.g., kaolin and bentonite); emulsifiers; preservatives;sweeteners; stabilizers; coloring agents; perfuming agents; flavoringagents; lubricants (e.g., talc, calcium and magnesium stearate); solidpolyethyl glycols; and mixtures thereof.

The terms “pharmaceutically or cosmetically acceptable carrier” or“pharmaceutically or cosmetically acceptable vehicle” are used herein tomean, without limitations, any liquid, solid or semi-solid, includingbut not limited to water or saline, a gel, cream, salve, solvent,diluent, fluid ointment base, ointment, paste, implant, liposome,micelle, giant micelle, and the like, which is suitable for use incontact with living animal or human tissue without causing adversephysiological or cosmetic responses, and which does not interact withthe other components of the composition in a deleterious manner. Otherpharmaceutically or cosmetically acceptable carriers or vehicles knownto one of skill in the art may be employed to make compositions fordelivering the molecules of the present invention.

The formulations can be so constituted that they release the activeingredient only or preferably in a particular location, possibly over aperiod of time. Such combinations provide yet a further mechanism forcontrolling release kinetics. The coatings, envelopes, and protectivematrices may be made, for example, from polymeric substances or waxes.

Methods of in vivo administration of the compositions of the presentinvention, or of formulations comprising such compositions and othermaterials such as carriers of the present invention that areparticularly suitable for various forms include, but are not limited to,oral administration (e.g. buccal or sublingual administration), analadministration, rectal administration, administration as a suppository,topical application, aerosol application, inhalation, intraperitonealadministration, intravenous administration, transdermal administration,intradermal administration, subdermal administration, intramuscularadministration, intrauterine administration, vaginal administration,administration into a body cavity, surgical administration at thelocation of a tumor or internal injury, administration into the lumen orparenchyma of an organ, and parenteral administration. Techniques usefulin the various forms of administrations above include but are notlimited to, topical application, ingestion, surgical administration,injections, sprays, transdermal delivery devices, osmotic pumps,electrodepositing directly on a desired site, or other means familiar toone of ordinary skill in the art. Sites of application can be external,such as on the epidermis, or internal, for example a gastric ulcer, asurgical field, or elsewhere.

The compositions of the present invention can be applied in the form ofcreams, gels, solutions, suspensions, liposomes, particles, or othermeans known to one of skill in the art of formulation and delivery oftherapeutic and cosmetic compounds. Ultrafine particle sizes ofelectroprocessed materials can be used for inhalation delivery oftherapeutics. Some examples of appropriate formulations for subcutaneousadministration include but are not limited to implants, depot, needles,capsules, and osmotic pumps. Some examples of appropriate formulationsfor vaginal administration include but are not limited to creams andrings. Some examples of appropriate formulations for oral administrationinclude but are not limited to: pills, liquids, syrups, and suspensions.Some examples of appropriate formulations for transdermal administrationinclude but are not limited to gels, creams, pastes, patches, sprays,and gels. Some examples of appropriate delivery mechanisms forsubcutaneous administration include but are not limited to implants,depots, needles, capsules, and osmotic pumps. Formulations suitable forparenteral administration include but are not limited to aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets commonly used by oneof ordinary skill in the art.

Embodiments in which the compositions of the invention are combinedwith, for example, one or more “pharmaceutically or cosmeticallyacceptable carriers” or excipients may conveniently be presented in unitdosage form and may be prepared by conventional pharmaceuticaltechniques. Such techniques include the step of bringing intoassociation the compositions containing the active ingredient and thepharmaceutical carrier(s) or excipient(s). In general, the formulationsare prepared by uniformly and intimately bringing into association theactive ingredient with liquid carriers. Preferred unit dosageformulations are those containing a dose or unit, or an appropriatefraction thereof, of the administered ingredient. It should beunderstood that in addition to the ingredients particularly mentionedabove, formulations comprising the compositions of the present inventionmay include other agents commonly used by one of ordinary skill in theart. The volume of administration will vary depending on the route ofadministration. For example, intramuscular injections may range involume from about 0.1 ml to 1.0 ml.

The compositions of the present invention may be administered to personsor animals to provide substances in any dose range that will producedesired physiological or pharmacological results. Dosage will dependupon the substance or substances administered, the therapeutic endpointdesired, the desired effective concentration at the site of action or ina body fluid, and the type of administration. Information regardingappropriate doses of substances are known to persons of ordinary skillin the art and may be found in references such as L.S. Goodman and A.Gilman, eds, The Pharmacological Basis of Therapeutics, MacmillanPublishing, New York, and Katzung, Basic & Clinical Pharmacology,Appleton & Lang, Norwalk, Conn., (6^(th) Ed. 1995). One desirable dosagerange is 0.01 μg to 100 mg. Another desirable dosage range is 0.1 μg to50 mg. Another desirable dosage range is 0.1 μg to 1.0 μg. A clinicianskilled in the art of the desired therapy may chose specific dosages anddose ranges, and frequency of administration, as required by thecircumstances and the substances to be administered. For example, aclinician skilled in the art of hormone replacement therapy may chosespecific dosages and dose ranges, and frequency of administration, for asubstance such as progesterone, to be administered in combination withthe estrogenic and estrogenic modulatory molecules as required by thecircumstances. For example, progesterone, and other progestins known toone of skill in the art may be administered in amounts ranging fromabout 50 μg to 300 mg, preferably 100 μg to 200 mg, more preferably 1 mgto 100 mg. Specific dosages and combinations of dosages of estrogenicand estrogenic modulatory molecules and progestins will depend on theroute and frequency of administration, and also on the condition to betreated. For example, when the composition is formulated for oraladministration, preferably in the form of a dosage unit such as acapsule, each dosage unit may preferably contain 1 μg to 5 mg ofestrogenic and estrogenic modulatory molecules and 50 μg to 300 mg ofprogesterone. U.S. Pat. No. 4,900,734 provides additional examples ofacceptable dose combinations of estrogenic molecules and progestins.

Other Uses Involving Electrically or Magnetically Active Constituents

The compositions of the present invention have a number of additionaluses aside from substance delivery. Embodiments exist in which theincorporation of electrically or magnetically active constituents in theelectroprocessed material allows the electroprocessed material to moverhythmically in response to an oscillating electric or magnetic field.Such an electroprocessed material can be used, for example, in a leftventricular assist device by providing a pumping action or a ventricularmassage to a heart patient. Oscillations can be accomplished by passivemovement of a magnetic or electric field with respect to the conductivematerial, or vice versa. By manipulating material selection, theelectroprocessed material can be designed to remain in place permanentlyor to dissolve over time, eliminating the need for surgery to recoverthe device once the heart had recovered sufficiently.

Embodiments also exist in which an implanted electroprocessed materialis used to convey an electric charge or current to tissue. For example,electrically active constituents can be electrically stimulated topromote neural ingrowth, stem cell differentiation, or contraction ofengineered muscle, or to promote the formation of bone in orthopedicapplications in which electroprocessed material is used as a carrier toreconstruct bone. In one embodiment, for example, an electroprocessedmaterial is applied to a bone injury site and used to apply an electriccurrent to the material to facilitate and to promote healing. Theapplication of a small electric current to an injured bone is known toaccelerate healing or promote the healing of bone injuries.

In other embodiments involving magnetically reactive materials, amagnetic field is used to position an electroprocessed materialcontaining substances by relatively non-invasive means, for example bydirecting the movement of the material within the peritoneum. In otherembodiments, a composition containing electrically active compounds isused to produce electric field-driven cell migration. This approachaccelerates the healing process and minimize the risk of bacterialcolonization. In one example, an orthopedic implant is coated with avery thin (<100 microns) layer of an electrically active polymer. With avery thin electrode attached to the coating, upon post-implantation, anelectric field can be applied via an external electrode such that theelectric field-driven cell migration is towards the implant surface. Thedirection can be reversed if so desired. Field orientation depends onthe geometry of the implant and external electrode.

Use in Gene Therapy

Compositions of the present invention are also useful for testing andapplying various gene therapies. By working with the compositions invitro, different types of gene therapy and manipulation can be achievedby inserting preselected DNA in suspensions of cells, materials, etc.For example, nonviral techniques such as electroporation are used totreat cultured cells prior to insertion into the matrix of the presentinvention. In other embodiments, cells are treated within the matrixbefore the composition is inserted into a recipient. In vitro genetransfer avoids the exposure of a recipient to viral products, reducesrisk of inflammation from residual viral particles and avoids thepotential for germ cell line viral incorporation. It avoids the problemof finding or engineering viral coats large enough to accept large genessuch as the one for Factor VIII (anti-hemophilic factor). However, invivo gene therapy is accomplished in some embodiments by, for example,incorporating DNA into the electroprocessed material as it is createdthrough the electroprocessing techniques of the present invention,whereby some DNA will be incorporated into the in vivo cells in contactwith the composition after application of the composition to therecipient. This is especially true of small gene sequences, such asantisense oligonucleotides.

Use of an Electroprocessed Composition as Tissue or Organ Replacement

The ability to combine cells in an electroprocessed material providesthe ability to use the compositions of the present invention to buildtissue, organs, or organ-like tissue. Cells included in such tissues ororgans can include cells that serve a function of delivering asubstance, seeded cells that will provide the beginnings of replacementtissue, or both. Many types of cells can be used to create tissue ororgans. Stem cells, committed stem cells, and/or differentiated cellsare used in various embodiments. Also, depending on the type of tissueor organ being made, specific types of committed stem cells are used.For instance, myoblast cells are used to build various musclestructures, neuroblasts are employed to build nerves, and osteoblastsare chosen to buildbone. Examples of stem cells used in theseembodiments include but are not limited to embryonic stem cells, bonemarrow stem cells and umbilical cord stem cells used to make organs ororgan-like tissue such as livers, kidneys, etc. Examples of tissueembodiments that use differentiated cells include fibroblasts in amatrix used for a patch, for example a hernia patch, endothelial cellsfor skin, osteoblasts for bone, and differentiated cells like cadaverdonor pancreatic islet cells for a delivery device to place these cellsin a specific site, for example the liver. In some embodiments the shapeof the electroprocessed composition helps send signals to the cells togrow and reproduce in a specific type of desired way. Other substances(for example, differentiation inducers) can be added to theelectroprocessed matrix to promote specific types of cell growth.Further, different mixtures of cell types are incorporated into thecomposition in some embodiments.

In certain disease states, organs are scarred to the point of beingdysfunctional. A classic example is cirrhosis. In cirrhosis; normalhepatocytes are trapped in fibrous bands of scar tissue. In oneembodiment of the invention, the liver is biopsied, viable liver cellsare obtained then cultured in an electroprocessed matrix, andre-implanted in the patient as a bridge to or replacement for routineliver transplantations.

Mixing of committed cell lines in a three dimensional electroprocessedmatrix can be used to produce structures that mimic complex organs. Forexample, by growing glucagon secreting cells, insulin secreting cells,somatostatin secreting cells, and/or pancreatic polypeptide secretingcells, or combinations thereof, in separate cultures, and then mixingthem together with electroprocessed materials through electroprocessing,an artificial pancreatic islet is created. These structures are thenplaced under the skin, retroperitoneally, intrahepatically or in otherdesirable locations, as implantable, long-term treatments for diabetes.

In other examples, hormone-producing cells are used, for example, toreplace anterior pituitary cells to affect synthesis and secretion ofgrowth hormone secretion, luteinizing hormone, follicle stimulatinghormone, prolactin and thyroid stimulating hormone, among others.Gonadal cells, such as Leydig cells and follicular cells are employed tosupplement testosterone or estrogen levels. Specially designedcombinations are useful in hormone replacement therapy in post andperimenopausal women, or in men following decline in endogenoustestosterone secretion. Dopamine-producing neurons are, used andimplanted in a matrix to supplement defective or damaged dopamine cellsin the substantia nigra. In some embodiments, stem cells from therecipient or a donor can be mixed with slightly damaged cells, forexample pancreatic islet cells, or hepatocytes, and placed in anelectroprocessed matrix and later harvested to control thedifferentiation of the stem cells into a desired cell type. Thisprocedure is performed in vitro or in vivo. The newly formeddifferentiated cells are introduced into the patient.

The ability to use electroprocessed materials and matrices tobioengineer tissue or organs creates a wide variety of bioengineeredtissue replacement applications. Examples of bioengineered componentsinclude, but are not limited to, skeletal muscle, cardiac muscle, nerveguides, brain constructs as a filler for damaged/removed areas of thebrain that are lost during accident or disease, a filler for othermissing tissues, cartilage scaffoldings, sheets for cosmetic repairs,skin (sheets with cells added to make a skin equivalent), vasculargrafts and components thereof, and sheets for topical applications (skincovering but no additional cells, just a patch). In some embodiments,such matrices are combined with drug and substance deliveryelectroprocessed matrices of the present invention in ways that willimprove the function of the implant. For example, antibiotics,anti-inflammatories, local anesthetics or combinations thereof, can beadded to the matrix of a bioengineered organ to speed the healingprocess and reduce discomfort.

One method or preparing implants of the present invention is use of abioreactor. There are several kinds of commercially availablebioreactors, devices designed to provide a low-shear, high nutrientperfusion environment. Until recently, most of the available bioreactorsmaintained cells in suspension and delivered nutrients and oxygen bysparging, through the use of impellers, or other means of stirring. TheRCCS bioreactor (Synthecon) is a rotating wall bioreactor. It consistsof a small inner cylinder, the substrate for the electrospinningprocess, positioned inside a larger outer cylinder. Although theelectrospun or electroaerosol matrix can be fabricated on the innercylinder, other locations within the bioreactor also can be used forplacement of a matrix for seeding. The gap between the inner and outercylinders serves as the culture vessel space for cells. Culture mediumis oxygenated via an external hydrophobic membrane. The low shearenvironment of the Synthecon RCCS bioreactor promotes cell-cell andcell-extracellular matrix (ECM) interactions without the damage or“washing away” of nutrients that occurs with active stirring orsparging. Typically, the RCCS device is operated at rotation rates of 8up to 60 RPM, as required to maintain cells in suspension, and at lessthan 8 RPM (preferably 2-3 RPM) for cultures immobilized along thecenter shaft of the vessel. The Synthecon bioreactor can be used in astandard tissue culture incubator. These values for spin rates and otherparameters can be varied depending on the specific tissue created.

Electroprocessed materials, such as matrices, are useful in formation ofprostheses. One application of the electroprocessed matrices is in theformation of medium and small diameter vascular prostheses. Somepreferred materials for this embodiment are collagen and elastin,especially collagen type I and collagen type III. Some examples include,but are not limited to coronary vessels for bypass or graft, femoralartery, popliteal artery, brachial artery, tibial artery, radial arteryor corresponding veins. The electroprocessed material is usefulespecially when combined with endothelial cells on the inside of thevascular prosthesis, and smooth muscle cells, for example a collagentube, and also when combined with fibroblasts on the outside of thecollagen tube. More complicated shapes including tapered and/or branchedvessels can also be constructed. A different-shaped mandrel is necessaryto wind the large fibers around or to orient theelectrospun/electroaerosol polymer.

Combination of electroprocessed matrix materials and wound polymerfibers can provide optimal growth conditions for cells. The polymerforms a basic structural matrix and the electroprocessed matrix is usedto deliver the cells. This facilitates cell attachment onto thestructural matrix. Furthermore the stress in the polymer also orientsfibers in the matrix providing further spatial cues for the cells.

In an alternative fabrication strategy, a cylindrical construct iselectrospun onto a suitable target, for example a cylindrical mandrel.Other shapes can be used if desirable based upon the shape of the siteinto which the implant will be placed. Matrices in this embodiment arecomposed, for example, of electroprocessed fibrinogen/fibrin (forexample to promote neovascularization, cellular integration andinfiltration from the surrounding tissue), electroprocessed collagen (topromote cell infiltration and lend mechanical integrity), and othercomponents, for example PGA, PLA, and PGA-PLA blends, PEO, PVA or otherblends. The relative ratio of the different components of this constructis tailored to specific applications (e.g. more fibrin, less collagenfor enhanced vascularization in a skin graft). To fabricate acylindrical muscle the construct is filled with muscle or stem cells orother cell type and the distal ends of the electrospun constructs aresutured or sealed shut. In some embodiments, cells are mixed withvarious matrix materials to enhance their distribution within theconstruct. For example, the cells can be mixed with electroprocessedfibrin or collagen prior to insertion into the construct. The objectiveof this strategy is to provide additional mechanical support to theconstruct and provide the cells with a three dimensional matrix withinthe construct to promote growth. This also helps to maintain the cellsin an even distribution within the construct. This method can be used toenhance the alignment of the cells within the construct. This fillingmaterial can be extruded directly into the cylindrical construct, as thefilling is extruded, alignment occurs. Mixing endothelial cells with theother cells inserted into the construct (or other cell types) can bedone to accelerate neovascularization. Another method to accomplish thisobjective is to electrodeposit endothelial cells directly into theelectroprocessed collagen-matrix that aids in formation of thecylindrical sheath. The alignment of the fibers within theelectroprocessed matrix that comprises the construct are optionallycontrolled by controlling the relative movement of the target and sourcesolution with respect to one another. Other cell types, such as tendonfibroblasts, are optionally electrospun into or onto the outer surfaceof the construct to enhance the formation of the outer connective tissuesheath that forms the construct.

In another example a sheet of electroprocessed material is prepared,rolled into a cylinder and inserted into an electroprocessed cylinder.The construct is filled with cells as described above, sutured shut andplaced in a bioreactor or directly in situ. By aligning the fibrils ofthe electrospun sheet of material in parallel with the long axis of theouter cylinder a muscle-like, electroprocessed composition is produced.Cells in contact with the fibrils that are arrayed along the long axisof the sheet spread in parallel with the fibrils of the sheet, forming amuscle construct of cells arrayed and layered in a pattern oforganization similar to that present in vivo. The cylindrical tissueconstruct is then implanted or placed within a RCCS bioreactor. Rates ofrotation to maintain this type of construct in suspension range from4-20 rpm, depending upon the over mass of the tissue and the specificmaterials used to fabricate the outer cylinder.

Vascularization of the engineered tissue containing electroprocessedmatrix material will occur in situ several days after surgery. In someembodiments, neovascularization of an engineered construct containingelectroprocessed material is enhanced by mixing endothelial cells intothe construct during fabrication. Another alternative for supplyingengineered tissue containing electroprocessed material with a vascularsupply is to temporarily transplant the tissue into the omentum. Theomentum has an extensive and rich vascular supply that can be used likea living incubator for the support of engineered tissue. The engineeredtissue is removed from a bioreactor, wrapped in the omentum andsupported by the diffusion of nutrients and oxygen from the surroundingtissue in the omentum. Alternatively, or in addition to this approach,engineered tissue is connected directly to the endogenous vascularsupply of the omentum. A blood vessel can be partially perforated or cutor left dissected free of the omentum. The engineered tissue containingelectroprocessed collagen, fibrin, or other materials, depending uponthe construct, is wrapped around the vessel. The engineered tissue issupported by nutrients leaking from the perforated vessel or by thesimple diffusion of nutrients if the vessel is left intact. Regardlessof strategy, the engineered tissue is surrounded by the omentum and itsrich vascular supply.

Tissue containing electroprocessed material can be engineered with anendogenous vascular system. This vascular system can be composed ofartificial vessels or blood vessels excised from a donor site on thetransplant recipient. The engineered tissue containing electroprocessedmatrix material is then assembled around the vessel. By enveloping sucha vessel with the tissue during or after assembly of the engineeredtissue, the engineered tissue has a vessel that can be attached to thevascular system of the recipient. In this example, a vessel in theomentum, or other tissue is cut, and the vessel of the engineered tissueis connected to the two free ends of the omental vessel. Blood passesfrom the omental vessel into the vascular system of the engineeredtissue, through the tissue and drains back into the omentum vessel. Bywrapping the tissue in the omentum and connecting it to an omental bloodvessel, the engineered tissue is supported by the diffusion of nutrientsfrom the omentum and the vessel incorporated into the tissue during itsfabrication. After a suitable period of time the tissue is removed fromthe omentum and placed in the correct site in the recipient. By usingthis strategy the engineered tissue containing electroprocessed materialis supported in a nutrient rich environment during the first severaldays following removal from the bioreactor. The environment of theomentum also promotes the formation of new blood vessels in implantedtissue. This omental incubator strategy can be combined with the otherstrategies such as combining angiogenic factors in the matrix materialduring electroprocessing. Several options are available. First, theimplants can be seeded with angioblasts and/or endothelial cells toaccelerate the formation of vascular elements once the engineered tissueis placed in situ. Second, angiogenic peptides can be introduced intothe engineered tissue via an osmotic pump. The use of an osmotic pumppermits delivery of peptides or, as noted, angiogenic peptides or growthfactors directly to the site of interest in a biologically efficient andcost-effective manner. VEGF delivered to ischemic hind limbs of rabbitsaccelerated capillary bed growth, increased vascular branching andimproved muscular performance with respect to ischemic controls. Analternative approach is to seed fully differentiated tissue constructscontaining electroprocessed matrix material with additional endothelialcells and or angioblasts shortly before they are implanted in situ.

In some embodiments, the stem cells or other cells used to construct theimplant are isolated from the subject, or other compatible donorrequiring tissue reconstruction. This provides the advantage of usingcells that will not induce an immune response, because they originatedwith the subject (autologous tissue) requiring the reconstruction.Relatively small biopsies can be used to obtain a sufficient number ofcells to construct the implant. This minimizes functional deficits anddamage to endogenous tissues that serve as the donor site for the cells.

In some embodiments, the matrices of the present invention includesubstances in the matrix that will improve the performance of theimplanted electroprocessed matrix. Examples of substances that can beused include peptide growth factors, antibiotics, and/or anti-rejectiondrugs. Alternatively, cells that are engineered to manufacture desiredcompounds can be included. The entire construct is, for example,cultured in a bioreactor or conventional culture or placed directly invivo. For example, neovascularization can be stimulated by angiogenicand growth-promoting factors, administered, as peptides, proteins or asgene therapy. Angiogenic agents can be incorporated into theelectroprocessed matrix. Nerve growth factors can be electrospun intothe matrix to promote growth or neurons into the matrix and tissue. In adegradable matrix, the gradual degradation/breakdown of the matrix willrelease these factors and accelerate growth of desired tissues.

Electroprocessed matrices can also be used in connection with othermatrix building processes. In other words, an extruded tube can have anoutside layer electrospun onto it wherein the different layerscomplement each other and provide an appropriate matrix to promote aspecific type of cell growth. As an example, a vascular graft comprisedprimarily of a collagen tube can have an electrospun layer of both othermaterials such as collagen or fibrin and cells added to promote theacceptability of the graft in a particular recipient. A second exampleis an in vitro skin preparation formed by growing fibroblasts in onelayer, covering the first layer with electroprocessed collagen, and thengrowing a second layer composed of epidermal cells in the fibrin matrix.This layering technique can be used to make a variety of tissues.

Stability and Storage of the Electroprocessed Compositions

The stability of the compositions of the present invention comprisingelectroprocessed materials combined with substances also allows for longterm storage of the compositions between formation and use. Stabilityallows greater flexibility for the user in embodiments in which a drugor other substance is applied after formation of the electroprocessedmaterial, for example by soaking and spraying. A formed electroprocessedmatrix can be fabricated and stored, and then the exact substancecomposition to be delivered to an individual patient can be prepared andtailored to a specific need shortly before implantation or application.This feature allows users greater flexibility in both treatment optionsand inventory management. Many electroprocessed materials are dry oncethey are spun, essentially dehydrated, thereby facilitating storage in adry or frozen state. Further, the electroprocessed compositions aresubstantially sterile upon completion, thereby providing an additionaladvantage in therapeutic and cosmetic applications.

Storage conditions for the compositions of the present invention willdepend on the electroprocessed materials and substances therein. Inembodiments involving proteins, for example, it may be necessary ordesirable to store the compositions at temperatures below 0° C., undervacuum, or in a lyophilized condition. Other storage conditions can beused, for example, at room temperature, in darkness, in vacuum or underreduced pressure, under inert atmospheres, at refrigerator temperature,in aqueous or other liquid solutions, or in powdered form. Persons ofordinary skill in the art recognize appropriate storage conditions forthe materials and substances contained in the compositions and will beable to select appropriate storage conditions.

The compositions of the present invention and formulations comprisingthose compositions may be sterilized through conventional means known toone of ordinary skill in the art. Such means include but are not limitedto filtration, radiation, and heat. The compositions the presentinvention may also be combined with bacteriostatic agents, such asthimerosal, to inhibit bacterial growth.

Formulations comprising the compositions of the present invention may bepresented in unit-dose or multi-dose containers, for example, sealedampules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules and tablets commonly used by one of ordinary skill inthe art. Preferred unit dosage formulations are those containing a doseor unit, or an appropriate fraction thereof, of the administeredingredient. It should be understood that in addition to the ingredientsparticularly mentioned above, the formulations of the present inventionmay include other agents commonly used by one of ordinary skill in theart.

The compositions of the present invention may be packaged in a varietyof ways depending upon the method used for administering thecomposition. Generally, an article for distribution includes a containerwhich contains the composition or a formulation comprising thecomposition in an appropriate form. Suitable containers are well-knownto those skilled in the art and include materials such as bottles(plastic and glass), sachets, ampules, plastic bags, metal cylinders,and the like. The container may also include a tamper-proof assemblageto prevent indiscreet access to the contents of the package. Inaddition, the container has deposited thereon a label which describesthe contents of the container. The label may also include appropriatewarnings.

The present invention is further illustrated by the following examples,which are not to be construed in any way as imposing limitations uponthe scope thereof. On the contrary, it is to be clearly understood thatresort can be had to various other embodiments, modifications, andequivalents thereof, which, after reading the description herein, cansuggest themselves to those skilled in the art without departing fromthe spirit of the present invention.

Example 1

Fibroblast growth factor (FGF, obtained from Chemicon, Temecula, Calif.)was dissolved in a solution of matrix material comprised of type Icollagen (80%), PGA (10%) and PLA (10%). The percentages refer to theweight of the materials with respect to one another. These materialswere dissolved in HFIP at a final concentration of 0.08 gm per ml.Sufficient FGF was added to 1 ml of solution to provide an FGFconcentration of 50 ng/ml of the collagen/PGA/PLA electrospinningsolution. The material was electrospun into the shape of a cylinder ontothe outer surface of a grounded and spinning 16 gauge needle about 25-35mm in length. After application, the cylinder was sutured shut looping asuture around the outside of the construct and pulling tight to seal theends. Alternatively, a hot forceps is used to pinch the ends togetherand literally heat seal the ends shut. These methods formed a hollow,enclosed construct. The construct was then surgically implanted withinthe vastus lateralis muscle of a rat. The construct was left in placefor seven days and recovered for inspection. FGF in the matrixaccelerated muscle formation within the electrospun matrix by promotingmuscle formation within the wall of the electrospun cylinder.

Example 2

Vascular endothelial growth factor (VEGF, obtained from Chemicon,Temecula, Calif.) was dissolved in a solution of matrix materialcomprised of type I collagen (80%), PGA (10%) and PLA (10%) as describedin EXAMPLE 1. These materials were dissolved in HFIP at a finalconcentration of 0.08 gm per ml. Sufficient VEGF was added to 1 ml ofsolution to provide a VEGF concentration of 50 ng/ml of thecollagen/PGA/PLA electrospinning solution. The material was electrospunto form a construct and implanted into a rat muscle using the sameprocedures set forth in Example 1. VEGF increased the density offunctional capillaries that were present throughout the construct. Thiswas evidenced by the presence of capillaries containing red blood cells(RBCs).

Example 3

Constructs of electroprocessed collagen and PGA:PLA copolymer, with VEGFspun into the matrix were prepared using 80% collagen and 20% PGA:PLA.The collagen and PGA:PLA were dissolved in HFIP at a final combinedconcentration of 0.08 gm per ml. Solutions were prepared in whichdifferent amounts of VEGF were added to 1 ml of the solution of collagenand PGA:PLA copolymer. Separate solutions were prepared containing 0.0ng, 25 ng, 50 ng, and 100 ng each in 1 ml. Constructs were prepared foreach solution by electrospinning one ml. The constructs were cut intosmaller sections and placed in a phosphate buffer solution (PBS).Release of VEGF into the PBS was measured as a function of time by theELISA method. The ELISA kit for VEGF was purchased from ChemiconInternational (part number cyt214) and the directions provided in thekit were followed to perform the ELISA. Samples were centrifuged toremove particulate matter and stored at −20° C. prior to use.

An identical construct was subjected to crosslinking by exposing it toglutaraldehyde vapor at room temperature and subjected to an identicalELISA assay. A sample of the electroprocessed construct was placed in a100 mm tissue culture dish. A 35 mm tissue culture dish containing 1 mlof 50% glutaraldehyde was placed inside the 100 mm tissue culture dish.The lid of the 100 mm tissue culture dish was replaced and the samplewas allowed to sit for 15 minutes at room temperature. The sample wasrinsed in sterile water or culture media. The amount of VEGF (expressedin picograms per 1 mg of electrospun material) for the non cross-linkedand cross-linked samples was measured at different times are presentedin FIGS. 3 and 4, respectively.

In FIG. 3 and FIG. 4, the open diamonds represent release from thefibers electrospun from the solution containing PGA:PLA copolymer andcollagen to which no VEGF was added. The open squares represent releasefrom fibers electrospun from the solution containing PGA:PLA copolymerand collagen to which 25 ng of VEGF were added. The open circlesrepresent release from the fibers electrospun from a solution containingPGA:PLA copolymer and collagen to which 50 ng of VEGF were added. Theopen triangles represent release from the fibers electrospun from asolution containing PGA:PLA copolymer and collagen to which 100 ng ofVEGF were added. Results demonstrate not only that the matrix releasesVEGF in PBS but also that cross-linking with glutaraldehyde slowsrelease from the matrix.

Example 4

Polyethylene-co-vinyl acetate) (PEVA) was a gift from DuPont (Elvax 40,40 vinyl acetate). PEVA pellets were soaked in ethanol for several daysto remove antioxidants. Poly(lactic acid) (100 L PLA) was a gift from byAlkermes, Inc. (Medisorb®) with a number-average molecular weight,M_(n), of 205 KD and polydispersity of 1.7. All solvents were analyticalgrade and were used as received. Tris(hydroxymethyl)aminomethanehydrochloride (Trizma® HCl) and trishydroxymethylaminomethane(Trizma®-base) were supplied by Sigma and were used without furtherpurification to prepare buffer solutions of pH 7.35. Tetracyclinehydrochloride was also obtained from Sigma. Actisite® periodontal fiber(0.5 nim PEVA) containing 25 wt % tetracycline hydrochloride was a giftfrom Alza Corporation (Palo Alto, Calif.).

Electrospinning was carried out using 14% wt/v solutions of 100% PEVA,100% PLA, or mixtures of the two in chloroform. The mixtures used were25% PEVA/75% PLA, 50%/50% of each, and 75% PEVA/25% PLA, withpercentages by weight. Tetracycline hydrochloride, which is insoluble inchloroform, was solubilized in a small amount of methanol and added tothe polymer solutions prior to electrospinning. The resulting solutionswere yellow but clear, indicating homogeneous solubilization of both thepolymers and drug.

The electrospinning set-up consisted of a glass pipette (held parallelto ground or angled at 45° downward), 0.32 mm diameter silver-coatedcopper wire (positive lead), a copper sheet (ground electrode) ca. 30 cmfrom the pipette, and a Spellman CZE100OR high voltage supply. Apositive voltage (15 kV) was applied through the copper wire to thepolymer solution inside the glass pipette. The solutions were deliveredvia syringe pumps to control the mass flow rate, which ranged from 10-18ml/h. More conveniently, the solution can be held in a plastic syringewith the high voltage supply connected to the metal syringe needle. Thesolutions were delivered via syringe pumps to control the mass flowrate, which ranged from 10-18 ml/h. The resulting electrically chargedfibers were collected on a rotating metal plate to produce a sheet ofnon-woven fabric.

A 100 L PLA containing 5% tetracycline hydrochloride (by weight) waselectrospun from 14% W/V solution in chloroform, with a mass flow rateof the polymer solution between 18-21 ml/h. PEVA containing 5%tetracycline hydrochloride (expressed herein as by weight of totalpolymer) was electrospun from 14% W/V solution with a mass flow rate of3 ml/h. Blends containing 5% tetracycline hydrochloride and consistingof 25% PLA and 75% PEVA were electrospun at mass flow rates of 13-18ml/h. A 50/50 PLA/PEVA blend with 5% tetracycline hydroxide was spun ata mass flow rate of 10-13 ml/h. A 50/50 PLA/PEVA blend with 25%tetracycline hydroxide (by weight of total polymer weight) was spun at amass flow rate of 15 ml/hr. A blend containing 75/25 PLA/PEVA with 5%tetracycline hydroxide was spun with a mass flow rate of 17 ml/h. Thecollected ‘fabric’ was used for studying the release of tetracyclinehydrochloride.

For comparative purposes, cast films were made from differentcompositions of PLA and PEVA. As with the fibers, films were made of 25%PEVA/75% PLA, 50%/50% of each, and 75% PEVA/25% PLA and 5% tetracyclinehydrochloride was added to each. The solutions in chloroform were castonto glass petri dishes, left at room temperature until the chloroformwas evaporated, then dried at 25° C. under vacuum for 3 hours. Therelease of tetracycline hydrochloride from ACTISITE® (PEVA) periodontalfiber was also compared.

Release of tetracycline hydrochloride was determined using UV-VISmeasurements carried out at Perkin-Elmer UV/VIS Lambda 40Spectrophotometer. The molar extinction coefficient for tetracyclinehydrochloride in Tris buffer was found to be 15,800 from a linearBeer-Lambert plot of absorbance at 360 nm vs. concentration. Release oftetracycline hydrochloride was determined by placing a known mass ofpolymer and drug in tris buffer and monitoring the absorbance at 360 nmas a function of time. The buffer solution was changed if the releaseddrug gave absorbance higher than 2.0. Data are reported as the %tetracycline hydrochloride released based upon the expected amount inthe samples from the feed composition. The morphology of the electrospunsamples were studied with JSM-820 Scanning electron microscope (JEOLLtd.).

The release profiles of tetracycline hydrochloride from electrospunfibers and the cast films are shown in FIGS. 5 and 6. In FIG. 5, thesolid diamonds denote release from the fibers electrospun from thesolution in which the polymer was 50% EVA and 50% PLA and 5%tetracycline was added. The open circles denote release from the fiberselectrospun from the solution in which the polymer was 50% EVA and 50%PLA and 25% tetracycline was added. The open triangles denote releasefrom the fibers electrospun from the solution in which the polymer was100% PLA and 5% tetracycline was added. The solid squares denote releasefrom the fibers electrospun from the solution in which the polymer was100% EVA and 5% tetracycline was added.

In FIG. 6, the open diamonds denote release from the fibers electrospunfrom the solution containing 100% EVA. The open squares denote releasefrom the ACTISITE® (PEVA) periodontal fiber. The open triangles denoterelease from the film in which the polymer was 50% PLA and 50% PEVA. Theopen circles denote release from the film in which the polymer was 25%PLA and 75% PEVA. The solid diamonds connected by a thick line denoterelease from the film containing 100% PLA. The solid triangles denoterelease from the film in which the polymer was 75% PLA and 25% PEVA. Thesolid squares denote release from the fibers electrospun from thesolution containing 25% PLA and 75% PEVA. The solid diamonds connectedby a thin line denote release from film containing 100% EVA.

Electrospun EVA showed a higher release rate than the mats derived fromPLA/EVA (50/50) or pure PLA. Electrospun PEVA released 65% of its drugcontent within 100 hours, whereas the electrospun 50/50 mixture of PEVAand PLA released about 40% over the same time period. Mats of PLA fiberswith no PEVA exhibit some instantaneous release, with negligible releaseover 50 hours. The 50:50 sample with 25 wt % tetracycline hydrochloridereleases the drug more rapidly than the 5% sample, although the %released of the former approaches that of the latter after 150 hrs.

FIG. 7 shows release profiles of three electrospun PEVA samples, twofrom the same batch of mat and another from a different preparationunder identical conditions. The release amounts of each sample aredenoted by solid diamonds, solid squares, and open triangles,respectively. The profiles are quite similar indicating very goodreproducibility. In general, the initial rate of release of allformulations including ACTISITE® (denoted as Alza) is high during thefirst 10-12 hours, most likely due to release of drug sequestered on thesample surfaces. The total percent released from the cast films (FIG. 6)were lower than that of the electrospun mats, as would be expected dueto the much lower surface area of the former. The PLA/EVA 75/25 filmreleased 30% of its tetracycline hydrochloride in 120 hrs, whereas thefilm of 50/50 PLA/EVA showed a slightly lower percent of release (25%)in the same period of time. Release from the PLA film was much lower,only 6% released in 120 hrs, whereas the PEVA film showed 8% releaseover the same period.

Example 5

A mixture of cultured insulin secreting cells is seeded into anelectroprocessed collagen matrix to form an electroprocessedcollagen-containing tissue. The electroprocessed matrix containing theinsulin secreting cells is implanted into a diabetic recipient in needof insulin. This electroprocessed collagen or fibrin-containing tissueoptionally contains a vessel. The matrix is implanted into theretroperitoneal space and the vessel is anastomosed into the hepaticportal circulation. Insulin is released from the insulin-containing celland transmitted to the circulation.

The electroprocessed matrix containing the insulin secreting cells isoptionally supplemented with cells that synthesize and secrete glucagon,somatostatin, pancreatic polypeptide, or combinations thereof, in orderto mimic the hormonal complement of the pancreatic islet.

Optionally, heterologous cells, (for example, engineered bacteria orcells from a conspecific donor) are placed in a matrix with a pore sizethat will allows diffusion of nutrients to the cells but does not allowor inhibits or delays the detection of the cells by the recipient'simmune system.

Example 6

Keratinocytes are harvested from a healthy site of a patient sufferingfrom a chronic wound. The cells are grown in culture and transfected byelectroporation to express VEGF. Next, the transfected cells are mixedor prepared in an electrospun collagen matrix. Antisense oligonucleotidefor matrix metalloproteinases (MMPs) are also spun into the matrix. Thematrix is topically applied to the surface of the wound. The cells nearand in the implant take up the antisense sequences, express theirtransfected gene sequences and MMP production is reduced. In otherapplications the cells may be genetically engineered to secrete VEGF,thereby promoting healing. Release of the antisense oligonucleotidessuppress expression of MMPs, which are typically overexpressed in achronic wound. Thus the wound site is repaired with an implant thatsimultaneously promotes natural healing responses. Optionally, thematrix is comprised of fibrin or a mix of fibrin and collagen. Thefibrin assists in cessation of bleeding and promotes healing.

Example 7

Osteoblasts from a patient with a bone injury are cultured andincorporated into an electrospun matrix comprising type I collagen. Thematrix is formed in the shape of a cavity or defect at the injury site.Bone growth factor (BGF), bone morphogenic protein (BMP) or sequences ofgenes encoding for these proteins, are electrospun into the matrix areoptionally incorporated into the electrospun matrix. The matrix assistsin growth of new bone, and the BGF or BMP in the matrix promotes bonegrowth.

Optionally, the collagen used is produced in vitro by geneticallyengineered cells that express a collagen polymer with more P-15 sitesthan in normal collagen. The excess of P-15 sites promotes osteoblaststo produce and secretes hydroxyapatite and further aid bone growth.

Optionally, the matrix is further electroprocessed with polypyrroles,which are electrically active materials. Electrodes are attached to eachend of the implanted matrix. Charged electrodes are later applied to thesurface over the electrodes to create a small electric current acrossthe implant to further facilitate healing of the bone injury. In anotherembodiment piezoelectric elements may be electrospun into the matrix toproduce electric discharges that promote healing.

Example 8

In another example, similar to that described for skeletal muscle acardiac patch is prepared. A sheet of electroprocessed material isprepared with aligned filaments of collagen. The sheet is folded into apleated sheet in the desired shape and or rolled into a cylinder. Asecond construct is electrospun in the desired shape, for example arectangle. The pleated sheet that mimics the cellular layers of theintact heart is inserted into the electroprocessed rectangular form. Theconstruct is filled with cells, sutured shut and placed in a bioreactoror directly in situ. By aligning the fibrils of the pleated electrospunsheet of material in parallel with the long axis of the outerrectangular form, a cardiac, muscle-like construct is obtained. Nativecardiac tissue is composed of layers of cells arrayed along a commonaxis with adjacent cell layers slightly off axis with the overlaying andunderlying layers. This structure is more precisely mimicked by themethods described below in which a matrix is prepared and cells aredirectly electroprocessed, dribbled or sprayed onto the matrix as it isprepared. Cells in contact with the fibrils that are arrayed along thelong axis of the sheet spread in parallel with the underlying fibrils ofthe sheet, forming a muscle construct of cells arrayed and layered in anin vivo-like pattern of organization. The construct can be directlyimplanted or placed within a RCCS bioreactor. Rates of rotation tomaintain this type of construct in suspension range from 4-20 rpm,depending upon the mass of the tissue and the specific materials used tofabricate the outer cylinder. Variations of this design include theaddition of angiogenic factors in the matrix, gene sequences, and agentsto suppress inflammation and/or rejection. Other cell types may be addedto the construct, for example microvascular endothelial cells, toaccelerate the formation of a capillary system within the construct.Other variations in this design principle can be used. For example,cells may be electroprocessed into the matrix as it is deposited on theground target. By varying the pitch of the fibers during spinning andspraying, dribbling or electroprocessing cells onto the fibers as theyare deposited very precisely controls the positioning of the cellswithin the construct.

All patents, publications and abstracts cited above are incorporatedherein by reference in their entirety. It should be understood that theforegoing relates only to preferred embodiments of the present inventionand that numerous modifications or alterations can be made thereinwithout departing from the spirit and the scope of the present inventionas defined in the following claims.

1. A composition comprising: an electroprocessed material and asubstance.
 2. The composition of claim 1, wherein the material is one ormore natural materials, one or more synthetic materials, or acombination thereof.
 3. The composition of claim 1, wherein the materialis a combination of one or more natural materials and one or moresynthetic materials.
 4. The composition of claim 2, wherein the naturalmaterial comprises one or more amino acids, peptides, denaturedpeptides, polypeptides, proteins, carbohydrates, lipids, nucleic acids,glycoproteins, lipoproteins, glycolipids, glycosaminoglycans,proteoglycans, or a combination thereof.
 5. The composition of claim 2,wherein the synthetic material comprises one or more polymers.
 6. Thecomposition of claim 1, wherein the substance comprises a therapeuticsubstance, a cosmetic substance or a combination thereof.
 7. Thecomposition of claim 1, wherein the substance comprises one or moremolecules, cells, objects, or combinations thereof.
 8. The compositionof claim 1, further comprising placement of the composition in abioreactor.
 9. The composition of claim 1, wherein the electroprocessedmaterial is crosslinked.
 10. A composition comprising anelectroprocessed material, wherein the electroprocessed materialprovides a biological effect when delivered to a desired location invivo or in vitro.
 11. A method of delivering a substance to a desiredlocation, comprising placing the composition of claim 1 at the desiredlocation.
 12. A method of delivering an electroprocessed material to adesired location, comprising placing the composition of claim 10 at thedesired location.
 13. The method of claim 11, wherein the substancecomprises a molecule or a cell that will release the molecule afterplacement in the desired location.
 14. The method of claim 11, whereinthe substance comprises a therapeutic substance, a cosmetic substance ora combination thereof.
 15. The method of claim 11, wherein the substancecomprises one or more molecules, cells, objects, or combinationsthereof.
 16. The method of claim 11, wherein the substance comprises acell.
 17. The method of claim 11, wherein the location is inside or uponthe body of a human or an animal.
 18. A method of manufacturing thecomposition of claim 1, comprising: electrodepositing one or moreelectrically-charged solutions comprising a material or moleculescapable of forming the material onto a grounded substrate underconditions effective to electrodeposit the material or the moleculescapable of forming the material on the substrate to form theelectroprocessed material; and, adding the substance to theelectrodeposited material to form the composition.
 19. The method ofclaim 18, wherein the material is one or more natural materials, one ormore synthetic materials, or a combination thereof.
 20. The method ofclaim 18, wherein the substance comprises a therapeutic substance, acosmetic substance or a combination thereof.
 21. The method of claim 18,wherein the substance comprises one or more molecules, cells, objects,or combinations thereof.
 22. A method of manufacturing the compositionof claim 10, comprising electrodepositing one or moreelectrically-charged solutions comprising a material or moleculescapable of forming the material onto a grounded substrate underconditions effective to electrodeposit the material or the moleculescapable of forming the material on the substrate to form theelectroprocessed material.
 23. The method of claim 22, wherein thematerial is one or more natural materials, one or more syntheticmaterials, or a combination thereof.